Homogenous antibody drug conjugates via enzymatic methods

ABSTRACT

The present application in one aspect provide Fc-containing polypeptide conjugates comprising an Fc-containing polypeptide conjugated to a conjugate moiety, wherein the Fc-containing polypeptide comprises an N-glycosylated Fc region comprising an acceptor glutamine residue flanked by an N-glycosylation site and wherein the conjugate moiety is conjugated to the Fc-containing polypeptide via the acceptor glutamine residue. Also provided are methods of making such Fc-containing polypeptide conjugates by using a wildtype or engineered transglutaminases. Further provided are engineered transglutaminases specifically designed for carrying out such reactions.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is divisional of U.S. patent application Ser. No.15/317,907, filed on Dec. 9, 2016, which is a National Phase applicationunder 35 U.S.C. § 371 of International Application No.PCT/US2015/035375, filed on Jun. 11, 2015, which claims the prioritybenefit of U.S. Provisional Application No. 62/011,534, filed on Jun.12, 2014, the disclosures of which are hereby incorporated by referencein their entirety.

SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE

The content of the following submission on ASCII text file isincorporated herein by reference in its entirety: a computer readableform (CRF) of the Sequence Listing (file name:720692000110SEQLISTING.txt, date recorded: May 22, 2018, size: 51 KB).

BACKGROUND

Antibody-based therapeutics have played an important role in targetedtherapy for various disorders, such as cancers and immunologicaldiseases. In recent years, antibody drug conjugates (ADC) have beenexplored extensively for effective delivery of drugs to target sites.For example, chemical modification has been widely used for conjugatingdrugs to antibodies either through lysine side chain amines or throughcysteine sulfhydryl groups. However, these conjugation methodsfrequently led to a heterogeneous mixture of conjugates having differentmolar ratios of drug to antibody, non-specific conjugation sites, aswell as different efficiency, safety, and pharmacokinetics. See Tanakaet al, FEBS Letters 579:2092-2096 (2005). Reactive cysteine residuesengineered at specific sites of antibodies for specific drug conjugationwith defined stoichiometry has also been made. See Junutula et al.,Nature Biotechnology, 26: 925-932 (2008). However, expression andconjugation of such cysteine engineered antibodies and antibody-drugconjugates require lengthy and complicated reaction procedures. See,e.g., Gomez et al., Biotechnology and Bioengineering, 105(4): 748-760(2009). Antibody aggregates may also be generated during the process ofmaking the cysteine engineered antibodies and the antibody-drugconjugates. Unnatural amino acid residues have also been incorporatedinto antibodies as chemical handles for site-specific conjugation. SeeAxupa et al., PNAS, 109: 16101-16106 (2012). To implement thismethodology, an orthogonal pair of amber suppressor tRNA andaminoacyl-tRNA synthetase has to be integrated into an expression hostfirst. Then, the mutant antibody can be expressed in this special hostwith medium supplement of the unnatural amino acid. This process is notonly time consuming, but also very low in antibody expression yield.

Recently, enzymatic approaches to making ADCs using a transglutaminasehave been explored. Transglutaminases (TGase) transfers a moiety havingan amine donor group to an acceptor glutamine residue throughtransglutamination. Full-length IgG antibodies of human isotype containa conserved glutamine residue at position 295 of the heavy chain (Q295).Because this glutamine residue is in close proximity to anN-glycosylation site (N297), it was generally believed that Q295 on thefull length antibody is inaccessible to TGase when the antibody isN-glycosylated. To allow TGase acting on full length antibodies, the Fcregion of the antibody was deglycosylated or mutated to remove theN-glycosylation site prior to the TGase-mediated conjugation. SeeWO2013/092998. Alternatively, glutamine-containing sequence “tags” havebeen inserted into the antibodies' light or heavy chains to provideacceptor glutamine sites. See WO2012059882. Hence, all currentsite-specific ADC technologies rely on engineered antibody mutants,which may result in potential immunogenicity and in vivo instability.There is a strong need for an efficient site-specific antibodyconjugation tool where intact antibody can be used directly.

All publications, patents, and patent applications cited herein arehereby incorporated by reference herein in their entirety.

SUMMARY OF THE INVENTION

The present invention in one aspect provides an Fc-containingpolypeptide conjugate comprising an Fc-containing polypeptidesite-specifically conjugated to a conjugate moiety, wherein theFc-containing polypeptide comprises an N-glycosylated Fc region, whereinthe N-glycosylated Fc region comprises an acceptor glutamine residueflanked by an N-glycosylation site, and wherein the conjugate moiety isconjugated to the Fc-containing polypeptide via the acceptor glutamineresidue.

In some embodiments, the acceptor glutamine residue is flanked by anN-glycosylation site at +2 position relative to the glutamine residue.In some embodiments, the N-glycosylated Fc region comprises the aminoacids 290 to 300 of an immunoglobulin heavy chain, wherein the numberingis according to the Kabat index. In some embodiments, the N-glycosylatedFc region is the Fc region of a naturally occurring immunoglobulin heavychain.

In some embodiments according to any one of the embodiments above, theimmunoglobulin is selected from the group consisting of IgG1, IgG2,IgG3, and IgG4. In some embodiments, the Fc-containing polypeptide is animmunoglobulin heavy chain. In some embodiments, the Fc-containingpolypeptide is a full length antibody. In some embodiments, the antibodyis a human or humanized antibody. In some embodiments, both heavy chainsof the antibody are conjugated to the conjugate moiety. In someembodiments, the acceptor glutamine residue is at position 295 and theN-glycosylation site is at position 297, wherein the numbering isaccording to the Kabat index.

In some embodiments according to any of the embodiments above, theconjugate moiety comprises an active moiety selected from the groupconsisting of: a moiety that improves the pharmacokinetic property ofthe Fc-containing polypeptide, a therapeutic moiety, and a diagnosticmoiety. In some embodiments, the active moiety is a toxin.

In some embodiments according to any of the embodiments above, theFc-containing polypeptide and the conjugate moiety are conjugated via alinker, such as a cleavage linker or a non-cleavable linker.

In some embodiments, there is provided a composition comprising any oneof the Fc-containing polypeptide conjugate described above, wherein atleast about 50% (for example at least about any of 60%, 70%, 80%, 90%,or 95%) of the Fc-containing polypeptide conjugates in the compositionis glycosylated in the Fc region. In some embodiments, at least about50% (for example at least about any of 60%, 70%, 80%, 90%, or 95%) ofthe Fc-containing polypeptide conjugates has the Fc-containingpolypeptide to conjugate moiety molar ratio of 1:1 or 1:2.

In one aspect, there is provided an antibody drug conjugate comprisingan antibody conjugated to a conjugation moiety via an endogenousacceptor glutamine residue on the antibody, wherein the antibody drugconjugate is glycosylated in the Fc region. In some embodiments, theantibody drug conjugate is N-glycosylated in the Fc region. In someembodiments, the antibody is a human antibody. In some embodiments, theantibody is a humanized antibody.

In some embodiments according to any one of the antibody drug conjugatesdescribed above, both heavy chains of the antibody are conjugated to theconjugate moiety.

In some embodiments according to any one of the antibody drug conjugatesdescribed above, the conjugate moiety comprises an active moietyselected from the group consisting of: a moiety that improves thepharmacokinetic property of the antibody, a therapeutic moiety, and adiagnostic moiety. In some embodiments, the active moiety is a toxin.

In some embodiments according to any one of the antibody drug conjugatesdescribed above, the antibody and the conjugate moiety are conjugatedvia a linker. In some embodiments, the linker is a cleavable linker. Insome embodiments, the linker is a non-cleavable linker.

In some embodiments, there is provided a composition comprising any oneof the antibody drug conjugates described above, wherein at least about50% (for example, at least about any of 60%, 70%, 80%, 90%, or 95%) ofantibody drug conjugates in the composition is glycosylated in the Fcregion. In some embodiments, at least 80% of the antibody drugconjugates in the composition has the antibody to conjugate moiety molarratio of 1:1 or 1:2.

In another aspect, there is provided a method of making an Fc-containingpolypeptide conjugate comprising an Fc-containing polypeptidespecifically conjugated to a conjugate moiety comprising: reacting theFc-containing polypeptide with the conjugate moiety in the presence of atransglutaminase under a condition that is sufficient to generate theFc-containing polypeptide conjugate, wherein the Fc-containingpolypeptide comprises an N-glycosylated Fc region, wherein theN-glycosylated Fc region comprises an acceptor glutamine residue flankedby an N-glycosylation site, and wherein the conjugate moiety isconjugated to the Fc-containing polypeptide via the acceptor glutamineresidue.

In some embodiments, there is provided a method of making anFc-containing polypeptide conjugate comprising an Fc-containingpolypeptide specifically conjugated to a conjugate moiety comprising asmall molecule handle and an active moiety comprising: a) reacting theFc-containing polypeptide with the small molecule handle in the presenceof a transglutaminase under a condition that is sufficient to generatean intermediate conjugate comprising an Fc-containing polypeptidespecifically conjugated to the small molecule handle, and b) couplingthe intermediate conjugate with an active moiety thereby obtaining theFc-containing polypeptide conjugate, wherein the Fc-containingpolypeptide comprises an N-glycosylated Fc region, wherein theN-glycosylated Fc region comprises an acceptor glutamine residue flankedby an N-glycosylation site, and wherein the conjugate moiety isconjugated to the Fc-containing polypeptide via the acceptor glutamineresidue. In some embodiments, the transglutaminase is a wildtypetransglutaminase having the amino acid sequence of SEQ ID NO:16. In someembodiments, the transglutaminase is an engineered transglutaminase,such as an engineered transglutaminase comprising an amino acid sequencehaving at least about 80% (for example, at least about 85%, 90%, 95%, or99%) identity to SEQ ID NO:16. In some embodiments, the molar ratio ofthe transglutaminase and the Fc-containing polypeptide is about 10:1 toabout 1:100. In some embodiments, the transglutaminase is immobilized ona solid support. In other embodiments, the Fc-containing polypeptide isimmobilized on a solid support.

In some embodiments according to any one of the methods described above,the acceptor glutamine residue is flanked by an N-glycosylation site at+2 position relative to the glutamine residue. In some embodiments, theN-glycosylated Fc region comprises the amino acids 290 to 300 of animmunoglobulin heavy chain, wherein the numbering is according to theKabat index. In some embodiments, the N-glycosylated Fc region is the Fcregion of a wildtype immunoglobulin heavy chain. In some embodiments,the immunoglobulin is selected from the group consisting of IgG1, IgG2,IgG3, and IgG4. In some embodiments, the Fc-containing polypeptide is animmunoglobulin heavy chain, such as a full length antibody, for examplea human antibody or a humanized antibody. In some embodiments, bothheavy chains of the antibody are conjugated to the conjugate moiety. Insome embodiments, the acceptor glutamine residue is at position 295 andthe N-glycosylation site is at position 297, wherein the numbering isaccording to the Kabat numbering.

In some embodiments according to any one of the methods described above,the conjugate moiety comprises an active moiety selected from the groupconsisting of: a moiety that improves the pharmacokinetic property ofthe Fc-containing polypeptide, a therapeutic moiety, and a diagnosticmoiety. In some embodiments, the active moiety is a toxin.

In another aspect, there is provided a method of making an antibody drugconjugate comprising contacting an antibody composition with theconjugate moiety in the presence of a transglutaminase under a conditionsufficient to generate the antibody drug conjugate, wherein at leastabout 50% (for example, at least about any of 60%, 70%, 80%, 90%, or95%) of the antibody in the composition is glycosylated in theFc-region, and wherein the conjugate moiety is conjugated to theendogenous acceptor glutamine residue on the antibody.

In another aspect, there is provided a method of making an antibody drugconjugate comprising antibody specifically conjugated to a conjugatemoiety comprising a small molecule handle and an active moietycomprising a) contacting an antibody composition with the small moleculehandle in the presence of a transglutaminase under a conditionsufficient to generate an intermediate conjugate comprising an antibodyspecifically conjugated to the small molecule handle, and b) contactingthe intermediate conjugate with an active moiety thereby obtaining theantibody drug conjugate, wherein at least about 50% (for example, atleast about any of 60%, 70%, 80%, 90%, or 95%) of the antibody in thecomposition is glycosylated in the Fc-region, and wherein the conjugatemoiety is conjugated to the endogenous acceptor glutamine residue on theantibody.

In some embodiments according to any one of the methods of making anantibody drug conjugate described above, the transglutaminase is awildtype transglutaminase. In some embodiments, the wildtypetransglutaminase has the amino acid sequence of SEQ ID NO:16.

In some embodiments according to any one of the methods of making anantibody drug conjugate described above, the transglutaminase is anengineered transglutaminase. In some embodiments, the engineeredtransglutaminase comprises an amino acid sequence having at least about80% (for example, at least about 85%, 90%, 95%, or 99%) identity to SEQID NO:16.

In some embodiments according to any one of the methods of making anantibody drug conjugate described above, the transglutaminase has apurity of at least about 90% (for example, at least about any of 95%,98%, or 99%).

In some embodiments according to any one of the methods of making anantibody drug conjugate described above, the molar ratio of thetransglutaminase and the antibody composition is about 10:1 to about1:10.

In some embodiments according to any one of the methods of making anantibody drug conjugate described above, the transglutaminase isimmobilized on a solid support.

In some embodiments according to any one of the methods of making anantibody drug conjugate described above, the antibody is immobilized ona solid support.

In some embodiments according to any one of the methods of making anantibody drug conjugate described above, the antibody is a human orhumanized antibody.

In some embodiments according to any one of the methods of making anantibody drug conjugate described above, the conjugate moiety comprisesan active moiety selected from the group consisting of: a moiety thatimproves the pharmacokinetic property of the antibody composition, atherapeutic moiety, and a diagnostic moiety. In some embodiments, theactive moiety is a toxin.

In another aspect, there are provided engineered transglutaminases. Insome embodiments, there is provided an engineered transglutaminasecapable of conjugating an Fc-containing polypeptide (such as anantibody) to a conjugate moiety, wherein the Fc-containing polypeptide(such as the antibody) comprises an N-glycosylated Fc region, whereinthe N-glycosylated Fc region comprises an acceptor glutamine residueflanked by an N-glycosylation site, wherein upon reaction the conjugatemoiety is conjugated to the Fc-containing polypeptide (such as theantibody) via the acceptor glutamine residue, and wherein theconjugation is at least about 10% (for example, at least about any of20%, 30%, 40%, 50% or more) more active than a wildtype transglutaminaseunder the same reaction conditions. In some embodiments, there isprovided an engineered transglutaminase comprising an amino acidsequence having at least about 80% (for example, at least about 85%,90%, 95%, or 99%) identity to SEQ ID NO:16, wherein the transglutaminasecomprises a deletion selected from the group consisting of: D1-E4;P244-P247; and N282-L285.

Further provided are methods of making Fc-containing polypeptideconjugates (such as antibody drug conjugates) by using the engineeredtransglutaminases described herein.

In some embodiments, there is provided a method of making an antibodydrug conjugate comprising an antibody specifically conjugated to aconjugate moiety comprising: contacting an antibody composition with theconjugate moiety in the presence of any one of the engineeredtransglutaminases described above under a condition sufficient togenerate the antibody drug conjugate, wherein the conjugate moiety isconjugated to the endogenous acceptor glutamine residue on the antibody.

In some embodiments, there is provided a method of making an antibodydrug conjugate comprising an antibody specifically conjugated to aconjugate moiety comprising a small molecule handle and an active moietycomprising: a) contacting an antibody composition with the smallmolecule handle in the presence of any one of the engineeredtransglutaminases described above under a condition sufficient togenerate an intermediate conjugate comprising an antibody specificallyconjugated to the small molecule handle, and b) contacting theintermediate conjugate with an active moiety thereby obtaining theantibody drug conjugate, wherein the conjugate moiety is conjugated tothe endogenous acceptor glutamine residue on the antibody.

It is to be understood that one, some, or all of the properties of thevarious embodiments described herein may be combined to form otherembodiments of the present invention. These and other aspects of theinvention will become apparent to one of skill in the art.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 provides sequence alignments of the CH2 domain sequences ofvarious types of human, mouse, and rat IgGs. The endogenous glutamine(Q295) for TGase-mediated reaction and the N-glycosylation site (N297)are boxed.

FIG. 2 provides the alignment of amino acid sequence of TGases fromStrep Ladakanum (TG_SL, SEQ ID NO:16) and Strep Mobaraensis (TG_SM, SEQID NO:18). The consensus sequence is SEQ ID NO:19.

FIG. 3 provides sequences of deletion mutants based on TGases from StrepLadakanum. The sequence of a recombinant wildtype TG_SL is shown (SEQ IDNO:17).

FIG. 4 provides a diagram showing a one-step antibody-drug conjugationmethod.

FIG. 5 provides a diagram showing a two-step antibody-drug conjugationmethod.

FIG. 6 provides HPLC chromatograms for IgG1, 2 and 4 conjugated withMDC. FIG. 6, panel A shows conjugation of only the heavy chain of IgG1with MDC. FIG. 6, panel B shows conjugation of IgG1 with MDC in a molarratio of 1:1 and 1:2. FIG. 6, panel C shows site specific conjugation ofIgG2 (left) and IgG4 (right) with MDC.

FIG. 7 provides SDS PAGE analysis of IgG1-MDC conjugates.

FIG. 8 provides a maytansine derivative containing an extended,non-cleavable linear PEG linker with a primary amine group of molecularweight 896.42 Da, referred herein as MAY-PEG4.

FIG. 9 provides a maytansine derivative containing a cleavable linkerwith a self-immolative spacer and terminal lysine of molecular weight1224.58 Da, referred herein as MAY-PVCL.

FIG. 10 provides MALDI-TOF spectra for DAR0 (i.e. naked IgG1, toppanel), DAR 1 (middle panel) and DAR 2 (bottom panel) of IgG1-MAY-PEG4.

FIG. 11 provides MALDI-TOF spectra of naked IgG1 (left panel) and IgG1conjugated to MAY-PVCL (right panel).

FIG. 12 provides monomethyl auristatin E (MMAE) derivatives containing anon-cleavable linker with variable number of polyethylene glycol (PEG)units (top panel), referred herein as PEGx-MMAE, wherein x is 2, 4, 6,8, 10, 12, 16, 20 or 24; and an MMAE derivative containing a cleavablelinker (bottom panel), referred herein as PEG3c-MMAE.

FIG. 13 provides in vivo efficacy of trastuzumab-PEGx-MMAE conjugatesprepared by Tgase in BT474 xenograft mice.

FIG. 14 provides in vivo stability of a trastuzumab-PEG12-MMAE conjugateprepared by Tgase in NCI N87 xenograft mice.

FIG. 15 provides comparison of in vivo efficacy of atrastuzumab-PEG3c-MMAE conjugate (DAR2, referred herein as TP3cE) andTDM-1 (Genentech) conjugate in NCI N87 xenograft mice.

FIG. 16 provides comparison of in vivo stability of atrastuzumab-PEG3c-MMAE conjugate (DAR2, referred herein as TP3cE) andTDM-1 (Genentech) conjugate in NCI N87 xenograft mice.

FIG. 17 provides a comparison of in vivo efficacy of atrastuzumab-PEG3c-MMAE conjugate (DAR2, referred herein as TP3cE) andTDM-1 (Genentech) conjugate in SK_Ov3 xenograft mice. Arrows in the plotindicate time points for administration of the doses of antibody drugconjugates.

FIG. 18 provides a group of 3-arm PEG linkers (top panel; 1 to 5 k Da)each with one amine group and two azide groups, and Alkyne-PEG4c-MMAE(bottom panel) used in DAR4 ADC preparation.

DETAILED DESCRIPTION

The present application for the first time provides methods of attachinga conjugate moiety (such as a drug) to an intact, unmodified (e.g.,glycosylation configuration left unaltered) antibody in a site-specificand stoichiometric fashion. This is accomplished either by utilizing awildtype TGase under a specific reaction condition and/or through anengineered TGase that is specifically designed to carry outsite-specific conjugation at an endogenous glutamine residue in the Fcregion. The methods of the present application allow for the productionof a homogeneous site-specific and stoichiometric antibody drugconjugate which would offer superior PK profile, broad therapeuticindex, and optimal potency. The methods allow conjugation of a drug toan intact antibody without introducing mutations and/or deglycosylatingthe antibody, thus minimize immunogenicity introduced by such extrasteps of manipulations. The glycans on the intact antibody, whenpresent, protect the antibody from degradation, leading to more stableantibody-drug conjugates. As no manipulation of the antibody wasnecessary prior to the transglutamination reactions, the TGase-basedantibody conjugation methods described herein are significantly moreefficient than those reported previously.

Thus, the present application in one aspect provides Fc-containingpolypeptide conjugates (such as antibody drug conjugate) comprising anFc-containing polypeptide (such as antibody) conjugated to a conjugatemoiety, wherein the Fc-containing polypeptide (such as antibody)comprises an N-glycosylated Fc region comprising an acceptor glutamineresidue flanked by an N-glycosylation site, and wherein the conjugatemoiety is conjugated to the Fc-containing polypeptide (such as antibody)via the acceptor glutamine residue.

In another aspect, there are provided methods of making Fc-containingpolypeptide conjugates (such as antibody drug conjugates) by using awildtype or engineered transglutaminase.

Further provided are engineered transglutaminases specifically designedfor carrying out such reactions.

Definitions

“Transglutaminase,” used interchangeably herein with “TGase,” refers toan enzyme capable of carrying out tranglutamination reactions. The term“transglutamination” as used herein refers to a reaction where theγ-glutaminyl of an acceptor glutamine residue from a protein/peptide istransferred to an amine group, such as a primary amine or the ε-aminogroup of lysine.

The term “acceptor glutamine residue,” when referring to an amino acidresidue of a polypeptide or protein, refers to a glutamine residue that,under suitable conditions, is recognized by a TGase and can becrosslinked to a conjugate moiety comprising a donor amine group by aTGase through a reaction between the glutamine and a donor amine group(such as lysine or a structurally related primary amine such as aminopentyl group).

An “endogenous acceptor glutamine residue on an antibody” used hereinrefers to an acceptor glutamine residue in a naturally occurringantibody Fc region. Such endogenous acceptor glutamine residue istypically Q295 by the Kabat numbering and flanked by an N-glycosylationsite at Asn297 position.

“Fc-containing polypeptide” used herein refers to a polypeptide (e.g.,an antibody or an Fc fusion protein) comprising the Fc region of animmunoglobulin heavy chain. The term “polypeptide” used herein includesboth single polypeptide chain and multiunit polypeptides. For example,Fc-containing polypeptide can be a full length antibody (such as anintact antibody), or it can be a single chain of the full lengthantibody.

“Fc region” as used herein refers to the polypeptide comprising theconstant region of an antibody heavy chain excluding the first constantregion immunoglobulin domain. For IgG, the Fc region may compriseimmunoglobulin domains CH2 and CH3 and the hinge between CH1 and CH2.

“Full length antibody” as used herein refers to a molecule thatconstitutes the natural biological form of an antibody, includingvariable and constant regions. For example, in most mammals, includinghumans and mice, the full length antibody of the IgG isotype is atetramer and consists of two identical pairs of two immunoglobulinchains, each pair having one light and one heavy chain, each light chaincomprising immunoglobulin domains VL and CL, and each heavy chaincomprising immunoglobulin domains VH, CH1, CH2, and CH3. In somemammals, for example in camels and llamas, IgG antibodies may consist ofonly two heavy chains, each heavy chain comprising a variable domainattached to the Fc region.

By “amino acid modification” herein is meant an amino acid substitution,insertion, and/or deletion in a polypeptide sequence. By “amino acidsubstitution” or “substitution” herein is meant the replacement of anamino acid at a given position in a protein sequence with another aminoacid. A “variant” of a polypeptide refers to a polypeptide having anamino acid sequence that is substantially identical to a referencepolypeptide, typically a native or “parent” polypeptide. The polypeptidevariant may possess one or more amino acid substitutions, deletions,and/or insertions at certain positions within the native amino acidsequence.

“Conservative” amino acid substitutions are those in which an amino acidresidue is replaced with an amino acid residue having a side chain withsimilar physicochemical properties. Families of amino acid residueshaving similar side chains are known in the art, and include amino acidswith basic side chains (e.g., lysine, arginine, histidine), acidic sidechains (e.g., aspartic acid, glutamic acid), uncharged polar side chains(e.g., glycine, asparagine, acceptor glutamine, serine, threonine,tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine,valine, leucine, isoleucine, proline, phenylalanine, methionine),beta-branched side chains (e.g., threonine, valine, isoleucine) andaromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,histidine).

The term “protecting group” refers to a group that temporarily protectsor blocks, i.e., intended to prevent from reacting, a functional group,e.g., an amino group, a hydroxyl group, or a carboxyl group, during thetransformation of a first molecule to a second molecule.

The phrase “moiety that improves the pharmacokinetic properties” refersto a moiety that changes the pharmacokinetic properties of the moleculethat the moiety is attached to in such a way that a better therapeuticor diagnostic effect can be obtained. The moiety can for exampleincrease the water solubility, increase the circulation time, or reduceimmunogenicity.

The phrase “linker” refers to a structural element of a compound thatlinks one structural element of said compound to one or more otherstructural elements of said same compound.

As used herein, “treating” or “treatment” is an approach for obtainingbeneficial or desired results including clinical results. For purposesof this invention, beneficial or desired clinical results include, butare not limited to, one or more of the following: alleviating one ormore symptoms resulting from the disease, diminishing the extent of thedisease, stabilizing the disease (e.g., preventing or delaying theworsening of the disease), preventing or delaying the recurrence of thedisease, delaying or slowing the progression of the disease,ameliorating the disease state, providing a remission (partial or total)of the disease, decreasing the dose of one or more other medicationsrequired to treat the disease, delaying the progression of the disease,and/or increasing quality of life.

The term “individual” refers to a mammal and includes, but is notlimited to, human, bovine, horse, feline, canine, rodent, or primate. Insome embodiments, the individual is human.

It is understood that aspects and embodiments of the invention describedherein include “consisting of” and “consisting essentially of” aspectsand embodiments.

Reference to “about” a value or parameter herein includes (anddescribes) variations that are directed to that value or parameter perse. For example, description referring to “about X” includes descriptionof “X.” The term “about X-Y” used herein has the same meaning as “aboutX to about Y.”

As used herein and in the appended claims, the singular forms “a,” “or,”and “the” include plural referents unless the context clearly dictatesotherwise.

Fc-Containing Polypeptide Conjugates

The present application in one aspect provides an Fc-containingpolypeptide conjugate (such as an antibody drug conjugate) comprising anFc-containing polypeptide (such as an antibody) site-specificallyconjugated to a conjugate moiety. The Fc-containing polypeptide (such asantibody) comprises an N-glycosylated Fc region. The N-glycosylated Fcregion comprises an acceptor glutamine residue flanked by anN-glycosylation site, and the conjugate moiety is conjugated to theFc-containing polypeptide (such as antibody) via the acceptor glutamineresidue.

The Fc region of an immunoglobulin in some embodiments comprises part orall of the hinge region. In some embodiments the Fc-containingpolypeptide comprises the Fc region of a naturally occurringimmunoglobulin. In some embodiments, the Fc-containing polypeptidecomprising an Fc region of IgG1, IgG2, IgG3, IgG4 subtypes, or from IgA,IgE, IgD, or IgM. In some embodiments, the Fc region is from human IgG,and the Fc region is from an amino acid residue at position Glu216 orAla231 to the carboxyl-terminus thereof according to the Kabat numberingsystem.

In some embodiments, the Fc-containing polypeptide is an Fc-containingfusion polypeptide wherein one or more functional polypeptides are fusedto the Fc region. Such functional polypeptides include, but are notlimited to, the target-binding region of a receptor, an adhesionmolecule, a ligand, an enzyme, a cytokine, and a chemokine.

The Fc regions described herein can be N-glycosylated. For example, insome embodiments, the polysaccharide chain attached at theN-glycosylation site is at least about any of 1, 10, 20, 30, 40, 50, 60,70, 80, 90, or 100 units.

The N-glycosylation site flanks the acceptor glutamine residue to whichthe conjugate moiety is attached. The inventor has for the first timedemonstrated that, through methods described further herein, it ispossible to attach a conjugate moiety to an acceptor glutamine residueflanked by an N-glycosylation site in the Fc-region. In someembodiments, the N-glycosylation site and the acceptor glutamine residueare 5 or less amino acid residues apart. In some embodiments, theN-glycosylation site and the acceptor glutamine are 5, 4, 3, 2, or 1amino acids apart. In some embodiments, the N-glycosylation site and theacceptor glutamine are next to each other. In some embodiments, theacceptor glutamine residue is flanked by an N-glycosylation site at +2position relative to the glutamine residue. In some embodiments, theacceptor glutamine residue is flanked by an N-glycosylation site at +1,+2, +3, +4, or +5 position relative to the glutamine residue. In someembodiments, the acceptor glutamine residue is flanked by anN-glycosylation site at −1, −2, −3, −4, or −5 position relative to theglutamine residue.

Thus, in some embodiments, there is provided an Fc-containingpolypeptide conjugate comprising an Fc-containing polypeptidesite-specifically conjugated to a conjugate moiety, wherein theFc-containing polypeptide comprises an N-glycosylated Fc region, whereinthe N-glycosylated Fc region comprises an acceptor glutamine residueflanked by an N-glycosylation site, and wherein the conjugate moiety isconjugated to the Fc-containing polypeptide via the acceptor glutamineresidue.

In some embodiments, there is provided an Fc-containing polypeptideconjugate comprising an Fc-containing polypeptide site-specificallyconjugated to a conjugate moiety, wherein the Fc-containing polypeptidecomprises an N-glycosylated Fc region, wherein the N-glycosylated Fcregion comprises an acceptor glutamine residue that is 5 or less aminoacids apart (including for example 4, 3, 2, or 1 amino acids part) fromthe N-glycosylation site, and wherein the conjugate moiety is conjugatedto the Fc-containing polypeptide via the acceptor glutamine residue.

In some embodiments, there is provided an Fc-containing polypeptideconjugate comprising an Fc-containing polypeptide site-specificallyconjugated to a conjugate moiety, wherein the Fc-containing polypeptidecomprises an N-glycosylated Fc region, wherein the acceptor glutamineresidue is flanked by an N-glycosylation site at +2 position relative tothe glutamine residue, and wherein the conjugate moiety is conjugated tothe Fc-containing polypeptide via the acceptor glutamine residue.

In some embodiments, there is provided an Fc-containing polypeptideconjugate comprising an Fc-containing polypeptide site-specificallyconjugated to a conjugate moiety, wherein the Fc-containing polypeptidecomprises an N-glycosylated Fc region, wherein the N-glycosylated Fcregion comprises amino acid sequence of SEQ ID NO:1 (KPREEQX₁NSTX₂R,wherein X₁ is Y or F and X₂ is Y or F), and wherein the conjugate moietyis conjugated to the Fc-containing polypeptide via the acceptorglutamine residue at position 6 of SEQ ID NO:1, and wherein theN-glycosylation is at position 8 of SEQ ID NO:1. In some embodiments,there is provided an Fc-containing polypeptide conjugate comprising anFc-containing polypeptide specifically conjugated to a conjugate moiety,wherein the Fc-containing polypeptide comprises an N-glycosylated Fcregion, wherein the N-glycosylated Fc region comprises amino acidsequence of SEQ ID NO:2 (KPREEQYNSTYR), and wherein the conjugate moietyis conjugated to the Fc-containing polypeptide via the acceptorglutamine residue at position 6 of SEQ ID NO:2, and wherein theN-glycosylation is at position 8 of SEQ ID NO:2.

In some embodiments, there is provided an Fc-containing polypeptideconjugate comprising an Fc-containing polypeptide site-specificallyconjugated to a conjugate moiety, wherein the Fc-containing polypeptidecomprises an N-glycosylated Fc region, wherein the N-glycosylated Fcregion comprises amino acid sequence of SEQ ID NO:3 (CH2 sequence ofhuman IgG1, see FIG. 1), and wherein the conjugate moiety is conjugatedto the Fc-containing polypeptide via the acceptor glutamine residue atposition 65 of SEQ ID NO:3, and wherein the N-glycosylation is atposition 67 of SEQ ID NO:3 (see residues in the box shown in FIG. 1).

In some embodiments, there is provided an Fc-containing polypeptideconjugate comprising an Fc-containing polypeptide specificallyconjugated to a conjugate moiety, wherein the Fc-containing polypeptidecomprises an N-glycosylated Fc region, wherein the N-glycosylated Fcregion comprises amino acid sequence of SEQ ID NO:4 (CH2 sequence ofhuman IgG2, see FIG. 1), and wherein the conjugate moiety is conjugatedto the Fc-containing polypeptide via the acceptor glutamine residue atposition 64 of SEQ ID NO:4, and wherein the N-glycosylation is atposition 66 of SEQ ID NO:4 (see residues in the box shown in FIG. 1).

In some embodiments, there is provided an Fc-containing polypeptideconjugate comprising an Fc-containing polypeptide specificallyconjugated to a conjugate moiety, wherein the Fc-containing polypeptidecomprises an N-glycosylated Fc region, wherein the N-glycosylated Fcregion comprises amino acid sequence of SEQ ID NO:5 (CH2 sequence ofhuman IgG3, see FIG. 1), and wherein the conjugate moiety is conjugatedto the Fc-containing polypeptide via the acceptor glutamine residue atposition 65 of SEQ ID NO:5, and wherein the N-glycosylation is atposition 67 of SEQ ID NO:5 (see residues in the box shown in FIG. 1).

In some embodiments, there is provided an Fc-containing polypeptideconjugate comprising an Fc-containing polypeptide specificallyconjugated to a conjugate moiety, wherein the Fc-containing polypeptidecomprises an N-glycosylated Fc region, wherein the N-glycosylated Fcregion comprises amino acid sequence of SEQ ID NO:6 (CH2 sequence ofhuman IgG4, see FIG. 1), and wherein the conjugate moiety is conjugatedto the Fc-containing polypeptide via the acceptor glutamine residue atposition 65 of SEQ ID NO:6, and wherein the N-glycosylation is atposition 67 of SEQ ID NO:6 (see residues in the box shown in FIG. 1).

In some embodiments, there is provided an antibody drug conjugatecomprising an antibody specifically conjugated to a conjugate moiety,wherein the antibody comprises an N-glycosylated Fc region, wherein theN-glycosylated Fc region comprises an acceptor glutamine residue flankedby an N-glycosylation site, and wherein the conjugate moiety isconjugated to the antibody via the acceptor glutamine residue. In someembodiments, the antibody is a human antibody. In some embodiments, theantibody is a humanized antibody. In some embodiments, the antibody is achimeric antibody. In some embodiments, the antibody is a bispecific ormultispecific antibody. In some embodiments, the antibody istrastuzumab.

In some embodiments, there is provided a full length antibody conjugatedto a conjugate moiety, wherein the full length antibody comprises anN-glycosylated Fc region, and wherein the conjugate moiety is conjugatedto the full length antibody via the acceptor glutamine residue atposition 295 of a heavy chains of the antibody, wherein the numbering isaccording to the EU index as in Kabat. In some embodiments, there isprovided an antibody conjugated to a conjugate moiety, wherein theantibody comprises an N-glycosylated Fc region, wherein the conjugatemoiety is conjugated to the antibody via the acceptor glutamine residueat position 295 of a heavy chains of the antibody, and wherein theN-glycosylation is at position 297 of the heavy chain, wherein thenumbering is according to the EU index as in Kabat.

In some embodiments, there is provided an antibody drug conjugatecomprising an antibody conjugated to a conjugation moiety via anendogenous acceptor glutamine residue on the antibody, wherein theantibody drug conjugate is glycosylated (for example N-glycosylated) inthe Fc region. In some embodiments, the antibody is a human antibody. Insome embodiments, the antibody is a humanized antibody. In someembodiments, the antibody is a chimeric antibody. In some embodiments,the antibody is a bispecific or multispecific antibody. In someembodiments, the antibody is trastuzumab.

In some embodiments, there is provided an antibody drug conjugatecomprising trastuzumab that is N-glycosylated in the Fc region, whereinthe trastuzumab is conjugated to a conjugation moiety via an endogenousacceptor glutamine residue flanked by the N-glycosylation site. In someembodiments, there is provided an antibody drug conjugate comprisingtrastuzumab that is N-glycosylated at position 297, wherein thetrastuzumab is conjugated to a conjugation moiety via an endogenousacceptor glutamine residue at position 295, wherein the numbering isaccording to the EU index as in Kabat.

In some embodiments, there is provided a composition comprising theFc-containing fusion polypeptide described herein, wherein at least some(but not necessarily all) of the Fc-containing fusion polypeptides inthe composition is glycosylated (for example N-glycosylated) in the Fcregion. For example, in some embodiments, there is provided acomposition comprising an antibody drug conjugate, wherein the antibodydrug conjugate comprises an antibody conjugated to a conjugation moietyvia an endogenous acceptor glutamine residue on the antibody, andwherein at least some of (for example at least about any of 50%, 60%,70%, 80%, 90%, or 95%) the antibody drug conjugates in the compositionis glycosylated (for example N-glycosylated) in the Fc region. In someembodiments, the antibody is a human antibody. In some embodiments, theantibody is a humanized antibody. In some embodiments, the antibody is achimeric antibody. In some embodiments, the antibody is a bispecific ormultispecific antibody. In some embodiments, the antibody istrastuzumab.

The conjugation methods described herein allow for the production ofFc-containing polypeptide conjugates (such as antibody drug conjugate)that are conjugated to a conjugate moiety in a specific andstoichiometrically controlled fashion. As used herein, the term“specifically conjugated” refers to the specific conjugation orcrosslinking of the conjugate moiety at a specific site of theFc-containing polypeptide (such as antibody), namely, the acceptorglutamine residue at the Fc region that is flanked by an N-glycosylationsite. Site specificity can be confirmed by various techniques,including, but not limited to, peptide mapping and protein sequencing.In some embodiments, the molar ratio of the conjugate moiety to theFc-containing polypeptide (such as antibody) on the Fc-containingpolypeptide conjugate (such as antibody drug conjugate) is about 1:1. Insome embodiments, the molar ratio of the conjugate moiety to theFc-containing polypeptide (such as antibody) on the Fc-containingpolypeptide conjugate (such as antibody drug conjugate) is about 2:1. Insome embodiments, at least about 80% (such as at least about any of 85%,90%, 95% or more) of the Fc-containing polypeptide conjugate (such asantibody drug conjugate) in the composition has the Fc-containingpolypeptide (such as antibody) to conjugate moiety molar ratio of about1:1. In some embodiments, at least about 80% (such as at least about anyof 85%, 90%, 95% or more) of the Fc-containing polypeptide conjugate(such as antibody drug conjugate) in the composition has theFc-containing polypeptide (such as antibody) to conjugate moiety molarratio of about 1:2. In some embodiments, at least about 80% (such as atleast about any of 85%, 90%, 95% or more) of the Fc-containingpolypeptide conjugate (such as antibody drug conjugate) in thecomposition has the Fc-containing polypeptide (such as antibody) toconjugate moiety molar ratio of about 1:1 or about 1:2.

The conjugate moiety described herein can be any moiety that can beconjugated to the acceptor glutamine residue, either directly or via asmall molecule handle as further described herein. The conjugationbetween the conjugation moiety and the acceptor glutamine residue iscarried out by conjugating the amine donor group of the conjugationmoiety or the small molecule handle to the acceptor glutamine residue.Thus, any conjugate moiety containing an amine donor group can bedirectly conjugated to the Fc-containing polypeptide. Any conjugatemoiety not containing an amine donor group can be indirectly conjugatedto the Fc-containing polypeptide via a small molecule handle whichcontains an amine donor group.

The term “amine donor group” as used herein refers to a reactive groupcontaining one or more reactive amines (e.g., primary amines). Forexample, the conjugate moiety can comprise an amine donor group (e.g.,primary amine —NH2), an optional linker, and an active moiety (e.g., asmall molecule). The conjugate moiety can also be a polypeptide or abiocompatible polymer containing a reactive Lys (e.g., an endogenousLys). The amine donor group in some embodiments is a primary amine(—NH2) that provides a substrate for transglutaminase to allowconjugation of the agent moiety to the Fc-containing polypeptide via theacceptor glutamine. Accordingly, the linkage between the donor glutamineand the amine donor group can be of the formula —CH₂—CH₂—CO—NH—.

In some embodiments, the Fc-containing polypeptide and the conjugatemoiety are linked through a linker. In some embodiments, the linker is anon-cleavable linker. Suitable non-cleavable linkers include, but arenot limited to, NH₂—R—X, NH₂NH—R—X, and NH₂—O—R—X, wherein R is alkyl orpolyethylene glycol group (also referred to as PEG), wherein X is theactive moiety. A polyethylene glycol group or PEG group may have aformula of —(CH₂CH₂O)_(n)—, wherein n is an integer of at least 1. Insome embodiments, n is any of 2, 4, 6, 8, 10, 12, 16, 20, or 24.

In some embodiments, the Fc-containing polypeptide and the conjugatemoiety are linked through a cleavable linker. Suitable cleavable linkersinclude, but are not limited to, Lys-Phe-X, Lys-Val-Cit-PABC-X,NH₂—(CH₂CH₂O)_(n)-Val-Cit-PABC-X, andNH₂—(CH₂CH₂O)_(n)-(Val-Cit-PABC-X)₂, wherein X is the active moiety, andn is an integer of at least 1 (such as any of 2, 4, 6, 8, 10, 12, 16,20, or 24). PABC refers to p-aminobenzyloxycarbonyl. Cit refers tocitrulline.

Other exemplary amine donor group-linkers include, but are not limitedto, Ac-Lys-Gly, aminocaproic acid, Ac-Lys-beta-Ala, amino-PEG2(Polyethylene Glycol)-C2, amino-PEG3-C2, amino-PEG6-C2, Ac-Lys-Val(valine)-Cit (citrulline)-PABC (p-aminobenzyloxycarbonyl),aminocaproyl-Val-Cit-PABC, putrescine, and Ac-Lys-putrescine.

In some embodiments, the conjugate moiety is linked to the acceptorglutamine residue via a —NH—(C)_(n)— linker, wherein the (C)_(n) is asubstituted or unsubstituted alkyl or heteroalkyl chain, wherein n is aninteger from about 1 to about 60. In some embodiments, the carbon of thechain is substituted with an alkoxyl, hydroxyl, alkylcarbonyloxy,alkyl-S—, thiol, alkyl-C(O)S—, amine, alkylamine, amide, or alkylamide.In some embodiments, n is about 2 to about 20.

In some embodiments, the linker is branched. In some embodiments, thelinker is linear. In some embodiments, the linker has more than one(such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, or more) attachment sites for the attachment of active moieties.These active moieties can be the same or different from each other. Forexample, the conjugate moiety may comprise a polyacetal- or polyacetalderivative-based polymer linked to a plurality of active moieties (suchas drug molecules).

In some embodiments, the conjugate moiety is selected from the groupconsisting of Alexa 488 cadaverine, 5-FITC cadaverine, Alexa 647cadaverine, Alexa 350 cadaverine, 5-TAMRA cadaverine, 5-FAM cadaverine,SR101 cadaverine, 5,6-TAMRA cadaverine, 5-FAM lysine,Ac(acetyl)-LysGly-MMAD (monomethyl auristatin D), Amino-PEG3(polyethylene glycol)-C2-MMAD, Amino-PEG6 C2-MMAD,Amino-PEG3-C2-amino-nonanoyl-MMAD,Aminocaproyl-Val(valine)-Cit(citrulline)-PABC(p-aminobenzyloxycarbonyl)-MMAD,Ac-Lys-Val-Cit-PABC-MMAD, Aminocaproyl-MMAD, Ac-Lys-beta-Ala-MMAD,amino-PEG2-C2-MMAE (monomethyl auristatin E), Aminocaproyl-MMAE,amino-PEG3-C2-MMAE, Aminocaproyl-MMAF (monomethyl auristatin F),Aminocaproyl-Val-Cit-PABC-MMAE, Aminocaproyl-Val-Cit-PABC-MMAF,putrescinyl-geldanamycin, and Ac-Lys-putrescinyl-geldanamycin. MMAErefers to monomethyl auristatin E or derivatives thereof.

In some embodiments, the conjugate moiety is a compound comprising adiamine. In some embodiments, the compound is selected from the groupconsisting of putrescine (butane-1,4-diamine), ethylenediamine,cadaverine (pentane-1,5-diamine), spermidine, spermine, hydrazine,1,3-diaminopropane, hexamethylenediamine, phenylenediamine,xylylenediamine, diphenylethylenediamine, 1,8-diaminonapthalene, andstereoisomers, isosteres, analogs or derivatives thereof.

In some embodiments, the conjugate moiety is a maytansine derivative,such as MAY-PEG4 shown in FIG. 8 or MAY-PVCL shown in FIG. 9.

In some embodiments, the conjugate moiety is an MMAE derivativecomprising a non-cleavable linker (such as an amino-(CH₂CH₂O)_(n)—linker, for example, PEGx-MMAE as shown in FIG. 12). In someembodiments, the conjugate moiety is an MMAE derivative comprising acleavable linker (such as PEG3c-MMAE shown in FIG. 12).

In some embodiments, there is provided an antibody drug conjugatecomprising trastuzumab that is N-glycosylated in the Fc region, whereinthe trastuzumab is conjugated to a conjugation moiety comprising atleast one MMAE (such as 1, 2, or more) through an acceptor glutamineresidue flanked by the N-glycosylation site. In some embodiments, theconjugation moiety is PEGx-MMAE as shown in FIG. 12, wherein x is aninteger selected from 2, 4, 6, 8, 10, 12, 16, 20, and 24. In someembodiments, the conjugation moiety is PEG3c-MMAE as shown in FIG. 12.In some embodiments, the conjugation moiety comprises two MMAE and a3-arm PEG linker.

In some embodiments, there is provided a composition comprising any ofthe antibody drug conjugated described above comprising trastuzumab. Insome embodiments, the average molar ratio between the active moiety(such as drug, e.g. MMAE) in the conjugation moiety to the trastuzumabin the composition is about any of 1:1, 2:1, or 4:1. In someembodiments, at least about 80% (such as at least about any of 85%, 90%,95% or more) of the antibody drug conjugate comprising trastuzumab inthe composition has a molar ratio between the active moiety (such asdrug, e.g. MMAE) in the conjugation moiety to the trastuzumab of about2:1. In some embodiments, at least about 80% (such as at least about anyof 85%, 90%, 95% or more) of the antibody drug conjugate comprisingtrastuzumab in the composition has a molar ratio between the activemoiety (such as drug, e.g. MMAE) in the conjugation moiety to thetrastuzumab of about 4:1.

In some embodiments, the Fc-containing polypeptide conjugate is presentin an individual (e.g., a mammal) at about 50% or more after at leastabout 1 day upon administration in vivo. In some embodiments, theFc-containing polypeptide conjugate) is present in an individual (e.g.,a mammal) at about any of 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, or 95% or more after at least about any of2 hours, 2-6 hours, 6-12 hours, 12-18 hours, 18-24 hours, 1 day, 2 days,3 days, 4 days, 5 days, 6 days, 1 week, or 2 weeks upon administrationin vivo.

Active Moieties

The conjugate moieties described herein in some embodiments comprise anactive moiety. In some embodiments, the conjugate moiety comprises anactive moiety that is a peptide or polypeptide. In some embodiments, theconjugate moiety comprises an active moiety that is a biocompatiblepolymer.

In some embodiments, the conjugate moiety comprises an active moietythat is a cytotoxic agent, an immunosuppressive agent, or an imagingagent (e.g., a fluorophore). In some embodiments, the cytotoxic agent isa chemotherapeutic agent. In some embodiments, the active a moiety isany one of: a moiety that improves the pharmacokinetic property of theFc-containing polypeptide, a therapeutic moiety, and a diagnosticmoiety. In some embodiments, the active moiety is a small molecule.

In some embodiments, the conjugation moiety comprises an active moietythat is a cytotoxic agent. Examples of a cytotoxic agent include, butare not limited to, an anthracycline, an auristatin, a dolastatin,CC-1065, a duocarmycin, an enediyne, a geldanamycin, a maytansine, apuromycin, a taxane, a vinca alkaloid, SN-38, tubulysin, hemiasterlin,and stereoisomers, isosteres, analogs or derivatives thereof. In someembodiments, the conjugation moiety comprises monodansylcadaverine(MDC). In some embodiments, the conjugation moiety comprises TAM1. Insome embodiments, the conjugation moiety comprises monomethyl auristatinE (MMAE).

The anthracyclines are derived from bacteria Streptomyces and have beenused to treat a wide range of cancers, such as leukemias, lymphomas,breast, uterine, ovarian, and lung cancers. Exemplary anthracyclinesinclude, but are not limited to, daunorubicin, doxorubicin (i.e.,adriamycin), epirubicin, idarubicin, valrubicin, and mitoxantrone.

Dolastatins and their peptidic analogs and derivatives, auristatins, arehighly potent antimitotic agents that have been shown to have anticancerand antifungal activity. See, e.g., U.S. Pat. No. 5,663,149 and Pettitet al., Antimicrob. Agents Chemother. 42:2961-2965 (1998). Exemplarydolastatins and auristatins include, but are not limited to, auristatinE, auristatin EB (AEB), auristatin EFP (AEFP), MMAD, MMAF, MMAE, and5-benzoylvaleric acid-AE ester (AEVB).

Duocarmycin and CC-1065 are DNA alkylating agents with cytotoxicpotency. See Boger and Johnson, PNAS 92:3642-3649 (1995). Exemplarydolastatins and auristatins include, but are not limited to,(+)-docarmycin A and (+)-duocarmycin SA, and (+)-CC-1065.

Enediynes are a class of anti-tumor bacterial products characterized byeither nine- and ten-membered rings or the presence of a cyclic systemof conjugated triple-double-triple bonds. Exemplary enediynes include,but are not limited to, calicheamicin, esperamicin, and dynemicin.

Geldanamycins are benzoquinone ansamycin antibiotic that bind to Hsp90(Heat Shock Protein 90) and have been used antitumor drugs. Exemplarygeldanamycins include, but are not limited to, 17-AAG(17-N-Allylamino-17-Demethoxygeldanamycin) and 17-DMAG(17-Dimethylaminoethylamino-17-demethoxygeldanamycin).

Maytansines or their derivatives maytansinoids inhibit cellproliferation by inhibiting the microtubules formation during mitosisthrough inhibition of polymerization of tubulin. See Remillard et al.,Science 189:1002-1005 (1975). Exemplary maytansines and maytansinoidsinclude, but are not limited to, mertansine (DM1) and its derivatives aswell as ansamitocin.

Taxanes are diterpenes that act as anti-tubulin agents or mitoticinhibitors. Exemplary taxanes include, but are not limited to,paclitaxel (e.g., TAXOL®) and docetaxel (TAXOTERE®).

Vinca alkyloids are also anti-tubulin agents. Exemplary vinca alkyloidsinclude, but are not limited to, vincristine, vinblastine, vindesine,and vinorelbine.

In some embodiments, the conjugate moiety comprises an active moietythat is an immunosuppressive agent. Examples of an immunosuppressiveagent include, but are not limited to, gancyclovier, etanercept,tacrolimus, sirolimus, voclosporin, cyclosporine, rapamycin,cyclophosphamide, azathioprine, mycophenolgate mofetil, methotrextrate,and glucocorticoid and its analogs.

In some embodiments, the conjugate moiety comprises an active moietythat is an imaging agent (e.g., a fluorophore), such as fluorescein,rhodamine, lanthanide phosphors, and their derivatives thereof. Examplesof fluorophores include, but are not limited to, fluoresceinisothiocyanate (FITC) (e.g., 5-FITC), fluorescein amidite (FAM) (e.g.,5-FAM), eosin, carboxyfluorescein, erythrosine, Alexa Fluor® (e.g.,Alexa 350, 405, 430, 488, 500, 514, 532, 546, 555, 568, 594, 610, 633,647, 660, 680, 700, or 750), carboxytetramethylrhodamine (TAMRA) (e.g.,5,-TAMRA), tetramethylrhodamine (TMR), and sulforhodamine (SR) (e.g.,SR101).

In some embodiments, the conjugate moiety comprises an active moietythat is a polypeptide. In some embodiments, the polypeptide is anantibody, such as a humanized, human, chimeric, or murine monoclonalantibody.

In some embodiments, the conjugate moiety comprises an active moietythat is a toxin polypeptide (or a toxin protein). Examples of a toxinpolypeptide include, but are not limited to, diphtheria A chain,nonbinding active fragments of diphtheria toxin, exotoxin A chain, ricinA chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordiiproteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII,and PAP-S), Momordica charantia inhibitor, curcin, crotin, Sapaonariaofficinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin,enomycin, tricothecenes, inhibitor cystine knot (ICK) peptides (e.g.,ceratotoxins), and conotoxin (e.g., KIIIA or SmIIIa).

In some embodiments, the conjugate moiety comprises a label such as aradioisotope. Examples of a radioisotope or other labels include, butare not limited to, 3H, 14C, 15N, 35S, 18F, 32P, 33P, 64Cu, 68Ga, 89Zr,90Y, 99Tc, 1231, 1241, 1251, 1311, 111In, 131In, 153Sm, 186Re, 188Re,211At, 212Bi, and 153Pb.

In some embodiments, the conjugate moiety comprises an active moietythat is selected from the group consisting of Alexa 488 cadaverine,5-FITC cadaverine, Alexa 647 cadaverine, Alexa 350 cadaverine, 5-TAMRAcadaverine, 5-FAM cadaverine, SR101 cadaverine, 5,6-TAMRA cadaverine,5-FAM lysine, Ac-Lys-Gly-MMAD, amino-PEG3-C2-MMAD, amino-PEG6-C2-MMAD,amino-PEG3-C2-amino-nonanoyl-MMAD], aminocaproyl-Val-Cit-PABC-MMAD,Ac-Lys-beta-Ala-MMAD, Aminocaproyl-MMAD, Ac-Lys-Val-Cit-PABC-MMAD,Aminocaproyl-MMAE, amino-PEG3-C2-MMAE, amino-PEG2-C2-MMAE,Aminocaproyl-MMAF, Aminocaproyl-Val-Cit-PABC-MMAE,Aminocaproyl-Val-Cit-PABC-MMAF, amino-PEG2-C2-MMAF, amino-PEG3-C2-MMAF,putrescinyl-geldanamycin, and Ac-Lys-putrescinyl-geldanamycin. In someembodiments, the amine donor agent is aminocaproyl-Val-Cit-PABC-MMAE,aminocaproyl-Val-Cit-PABC-MMAF, Ac-Lys-putrescinyl-geldanamycin,Ac-Lys-beta-Ala-MMAD, Ac-Lys-Val-Cit-PABC-MMAD,aminocaproyl-Val-Cit-PABC-MMAD, and amino-PEG6-C2-MMAD.

In some embodiments, the conjugate moiety comprises an active moietythat is a biocompatible polymer. The Fc-containing polypeptide can beconjugated to the biocompatible polymer to improve the biologicalcharacteristics of the Fc-containing polypeptide, e.g., to increaseserum half-life and bioactivity, and/or to extend in vivo half-lives.Examples of biocompatible polymers include water-soluble polymer, suchas polyethylene glycol (PEG) or its derivatives thereof andzwitterion-containing biocompatible polymers (e.g., a phosphorylcholinecontaining polymer).

Methods of Making Fc-Containing Polypeptide Conjugates

In another aspect, the present application provides methods of makingthe Fc-containing polypeptide conjugates (such as antibody drugconjugates) using wildtype or engineered transglutaminase.

The inventor has created engineered TGases that are designed tospecifically conjugate a conjugate moiety to an acceptor glutamineresidue on the Fc region of an Fc-containing polypeptide (such asantibody) that is flanked by an N-glycosylation site. Contrary toprevious belief that a glutamine residue on the Fc region flanked by anN-glycosylation site would be inaccessible to the action of TGase, theinventor has further surprisingly found that, by utilizing a specificreaction condition (for example a specific concentration of the enzyme),wildtype TGases are also able to conjugate a conjugate moiety to anacceptor glutamine residue on the Fc region that is flanked by anN-glycosylation site in a site-specific and stoichiometric manner.

The methods described herein in some embodiments involve a singleconjugation step. Such method is particularly suitable, for example,when a conjugation yield of between 20-98% is sufficient to generate asubstantial amount of the Fc-containing polypeptide conjugate. Theone-step method is also useful when the size of linker needs beminimized, when there is plenty of supply of the Fc-containingpolypeptide, when the drug solubility is moderate (for example about 100mg/L), and when time saving is a bigger concern than getting a highyield.

In some embodiments, the method involves two steps. First, a smallmolecule handle is conjugated to the Fc-containing polypeptide via aTGase to create an intermediate conjugate. Subsequently, an activemoiety is coupled to the intermediate conjugate via the small moleculehandle, either covalently or noncovalently. The small molecule handlecan be specifically designed to tailor the coupling of the activemoiety, thus allows the conjugation of any kind of active moiety to theFc-containing polypeptide. The two-step method is particularly usefulwhen the supply of the Fc-containing polypeptide and/or active moiety islimited, and when the active moiety (such as toxin) has low watersolubility and/or induces aggregation of the polypeptide. By using asmall molecule handle, the first enzymatic coupling step can allow theachievement of high yield in conjugation. The second chemoselectivecoupling step then only requires a reactant ratio of active moiety:Fc-containing polypeptide between 1.2 to 1.5. This may lead to a higheroverall conjugation yield than a one-step process.

Thus, in some embodiments, there is provided a method of making anFc-containing polypeptide conjugate comprising an Fc-containingpolypeptide specifically conjugated to a conjugate moiety comprising:contacting the Fc-containing polypeptide with the conjugate moiety inthe presence of a transglutaminase under a condition that is sufficientto generate the Fc-containing polypeptide conjugate, wherein theFc-containing polypeptide comprises an N-glycosylated Fc region, whereinthe N-glycosylated Fc region comprises an acceptor glutamine residueflanked by an N-glycosylation site, and wherein the conjugate moiety isconjugated to the Fc-containing polypeptide via the acceptor glutamineresidue. In some embodiments, there is provided a method of making anFc-containing polypeptide conjugate comprising an Fc-containingpolypeptide specifically conjugated to a conjugate moiety comprising:contacting a composition comprising Fc-containing polypeptides with theconjugate moiety in the presence of a transglutaminase under a conditionthat is sufficient to generate the Fc-containing polypeptide conjugate,wherein at least some (e.g., at least about 50%, 60%, 70%, 80%, 90%, ormore) the Fc-containing polypeptides comprise an N-glycosylated Fcregion, wherein the Fc region comprises an acceptor glutamine residueflanked by an N-glycosylation site, and wherein the conjugate moiety isconjugated to the Fc-containing polypeptide via the acceptor glutamineresidue.

In some embodiments, there is provided a method of making an antibodydrug conjugate comprising an antibody specifically conjugated to aconjugate moiety comprising: contacting the antibody with the conjugatemoiety in the presence of a transglutaminase under a condition that issufficient to generate the antibody drug conjugate, wherein the antibodyis glycosylated (e.g., N-glycosylated) in the Fc-region, and wherein theconjugate moiety is conjugated to the endogenous acceptor glutamineresidue on the antibody. In some embodiments, there is provided a methodof making an antibody drug conjugate comprising an antibody specificallyconjugated to a conjugate moiety comprising: contacting an antibodycomposition with the conjugate moiety in the presence of atransglutaminase under a condition sufficient to generate the antibodydrug conjugate, wherein at least about some (e.g., at least about 50%,60%, 70%, 80%, 90%, or more) of the antibody in the composition isglycosylated in the Fc-region, and wherein the conjugate moiety isconjugated to the endogenous acceptor glutamine residue on the antibody.

In some embodiments, there is provided a method of making anFc-containing polypeptide conjugate comprising an Fc-containingpolypeptide specifically conjugated to a conjugate moiety comprising asmall molecule handle and an active moiety comprising: a) contacting theFc-containing polypeptide with the small molecule handle in the presenceof a transglutaminase under a condition that is sufficient to generatean intermediate conjugate comprising an Fc-containing polypeptidespecifically conjugated to the small molecule handle, and b) contactingthe intermediate conjugate with an active moiety thereby obtaining theFc-containing polypeptide conjugate, wherein the Fc-containingpolypeptide comprises an N-glycosylated Fc region, wherein theN-glycosylated Fc region comprises an acceptor glutamine residue flankedby an N-glycosylation site, and wherein the conjugate moiety isconjugated to the Fc-containing polypeptide via the acceptor glutamineresidue. In some embodiments, there is provided a method of making anFc-containing polypeptide conjugate comprising an Fc-containingpolypeptide specifically conjugated to a conjugate moiety comprising asmall molecule handle and an active moiety comprising: a) contacting acomposition comprising Fc-containing polypeptides with the smallmolecule handle in the presence of a transglutaminase under a conditionthat is sufficient to generate an intermediate conjugate comprising anFc-containing polypeptide specifically conjugated to the small moleculehandle, and b) contacting the intermediate conjugate with an activemoiety thereby obtaining the Fc-containing polypeptide conjugate,wherein at least some (e.g., 50%, 60%, 70%, 80%, 90%, or more) theFc-containing polypeptides comprise an N-glycosylated Fc region, whereinthe Fc region comprises an acceptor glutamine residue flanked by anN-glycosylation site, and wherein the conjugate moiety is conjugated tothe Fc-containing polypeptide via the acceptor glutamine residue.

In some embodiments, there is provided a method of making an antibodydrug conjugate comprising an antibody specifically conjugated to aconjugate moiety comprising a small molecule handle and an active moietycomprising: a) contacting the antibody with the small molecule handle inthe presence of a transglutaminase under a condition that is sufficientto generate an intermediate conjugate comprising an antibodyspecifically conjugated to the small molecule handle, and b) contactingthe intermediate conjugate with an active moiety thereby obtaining theantibody drug conjugate, wherein the antibody is glycosylated (e.g.,N-glycosylated) in the Fc-region, and wherein the conjugate moiety isconjugated to the endogenous acceptor glutamine residue on the antibody.In some embodiments, there is provided a method of making an antibodydrug conjugate comprising antibody specifically conjugated to aconjugate moiety comprising a small molecule handle and an active moietycomprising: a) contacting an antibody composition with the smallmolecule handle in the presence of a transglutaminase under a conditionsufficient to generate an intermediate conjugate comprising an antibodyspecifically conjugated to the small molecule handle, and b) contactingthe intermediate conjugate with an active moiety thereby obtaining theantibody drug conjugate, wherein at least some (e.g., at least about anyof 50%, 60%, 70%, 80%, 90%, or more) of the antibody in the compositionis glycosylated (e.g., N-glycosylated) in the Fc-region, and wherein theconjugate moiety is conjugated to the endogenous acceptor glutamineresidue on the antibody.

The small molecule handle described herein generally has the structureof —NH₂—R, wherein R is a moiety that allows the attachment of theactivate moiety. The introduction of the small molecule handle in themethods described herein significantly increases the flexibility of themethods. Specifically, the structure of the small molecule handle can betailored to the attachment of the desired active moiety. For example, insome embodiments, R is a ligand which specifically binds to a bindingpartner. This allows attachment of any molecule (such as protein) thatcontains the binding partner. Suitable ligand/binding partner pairsinclude, but are not limited to, antibody/antigen, antigen/antibody,avidin/biotin, biotin/avidin, streptavidin/biotin, biotin/streptavidin,glutathione/GST, GST/glutathione, maltose binding protein/amylose,amylose/maltose binding protein, cellulose binding protein andcellulose, cellulose/cellulose binding protein, etc.

Other suitable small molecule handles described herein include, but arenot limited to, NH₂—CH₂—CH(OH)—CH₂—NH₂, NH₂—R—(OR′)₂, NH₂—R═O, NH₂—R—SH,NH₂-R-Azide. These small molecule handles allow the attachment of theconjugate moiety through suitable linkers such as NH₂—O—R—X,Maleimide-R—X, and Cyclooctyne-R—(R′—X)₂, wherein X is the activemoiety, and R and R′ are independently linker groups, such as linkergroups comprising alkyl or polyethylene glycol groups. In someembodiments, the small molecule handle is a 3-arm PEG linker with anamino group and two azide groups (such as the 3-arm PEG linker depictedin FIG. 18, top panel), wherein each of the azide groups may beconjugated to an active moiety.

The TGase-catalyzed reaction can be carried out from several hours to aday (e.g. overnight). The conjugate moiety or the small molecule handleare allowed to react with Fc-containing polypeptide (e.g., 1 mg/mL) atligand concentrations between 400 and 600 μmol/L, providing a 60 to90-fold excess of the substrates over the Fc-containing polypeptide, oroptionally at lower excess of substrates, e.g. 1- to 20-fold, or 10-20fold. The reactions can be performed in potassium-free phosphatebuffered saline (PBS; pH 8) at 37° C. After 4 h to several days,steady-state conditions are achieved. Excess ligand and enzyme are thenremoved using centrifugation-dialysis (VIVASPIN® MWCO 50 kDa,Vivascience, Winkel, Switzerland) or diafiltration (PELLICON® MWMCO 50kDa, Millipore). Reactions may be monitored by HPLC.

The resulting Fc-containing polypeptide conjugates can be analyzed usingany suitable method. For example, the stoichiometry of the conjugatedpolypeptide can be characterized by liquid chromatography massspectrometry (LC/MS) using a top-down approach in order to assess thenumber of conjugate moiety or small molecule handle conjugated toantibodies, and in particular the homogeneity of the composition.Conjugates can be reduced before LC/MS analysis and light chains andheavy chains are measured separately.

In one embodiment, the product is analyzed for drug loading (e.g. numberof active moiety in the conjugate per Fc-containing polypeptide). Suchmethods can be used to determine the mean number of conjugates or activemoieties (such as drug) per Fc-containing polypeptide as well as thedistribution of number of conjugates or active moieties (such as drug)per antibody in a composition, i.e. the percentage of total antibodywith any given level of drug loading or DAR. The portion of antibodieshaving a number (n) of conjugated acceptor glutamines (e.g. n=1, 2, 3,4, 5, 6, etc.) can be determined. One technique adapted to suchdetermination and more generally drug loading is hydrophobic interactionchromatography (HIC), HIC can be carried out as described for example inHamblen et al. (2004) Cancer Res. 10: 7063-7070; Wakankar et al. (2011)mAbs 3(2): 161-172; and Lyon et al (2012) Methods in Enzymology, Vol.502: 123-138, the disclosure of which are incorporated herein byreference.

The molar ratio between the transglutaminase and the Fc-containingpolypeptide in the conjugation reaction can be controlled to allowefficient transglutamination reaction. For example, in some embodiments,the molar ratio of the transglutaminase and the Fc-containingpolypeptide (such as antibody or antibody composition) is about 10:1 toabout 1:100, including any of about 10:1 to about 9:1, about 9:1 toabout 8:1, about 8:1 to about 7:1, about 7:1 to about 6:1, about 6:1 toabout 5:1, about 5:1 to about 4:1, about 4:1 to about 3:1, about 3:1 toabout 2:1, about 2:1 to about 1:1, about 1:1 to about 1:2, about 1:2 toabout 1:3, about 1:3 to about 1:4, about 1:4 to about 1:5, about 1:5 toabout 1:6, about 1:6 to about 1:7, about 1:7 to about 1:8, about 1:8 toabout 1:9, about 1:9 to about 1:10, about 1:10 to about 1:20, about 1:20to about 1:30, about 1:30 to about 1:40, about 1:40 to about 1:50, about1:50 to about 1:60, about 1:60 to about 1:70, about 1:70 to about 1:80,about 1:80 to about 1:90, or about 1:90 to about 1:100.

The amount of the transglutaminase in the reaction mixture can becontrolled to allow efficient transglutaminase reaction. For example, insome embodiments, the concentration of the tranglutaminase in thereaction mixture is about any of about 0.01 mg/ml to about 5 mg/ml,including for example any of about 0.01 mg/ml to about 0.02 mg/ml, about0.02 mg/ml to about 0.03 mg/ml, about 0.03 mg/ml to about 0.04 mg/ml,about 0.04 mg/ml to about 0.05 mg/ml, about 0.05 mg/ml to about 0.06mg/ml, about 0.06 mg/ml to about 0.07 mg/ml, about 0.07 mg/ml to about0.08 mg/ml, about 0.08 mg·ml to about 0.09 mg/ml, about 0.09 mg/ml toabout 0.1 mg/ml, about 0.1 mg/ml to about 0.2 mg/ml, about 0.2 mg/ml toabout 0.3 mg/ml, about 0.3 mg/ml to about 0.4 mg/ml, about 0.4 mg/ml toabout 0.5 mg/ml, about 0.5 mg/ml to about 0.6 mg/ml, about 0.6 mg/ml toabout 0.7 mg/ml, about 0.7 mg/ml to about 0.8 mg/ml, about 0.8 mg/ml toabout 0.9 mg/ml, about 0.9 mg/ml to about 1 mg/ml, about 1 mg/ml toabout 2 mg/ml, about 2 mg/ml to about 3 mg/ml, about 3 mg/ml to about 4mg/ml, about 4 mg/ml to about 5 mg/ml. In some embodiments, theconcentration of the transglutaminase in the reaction mixture is about0.05 mg/ml to about 1 mg/ml, such as about 0.2 mg/ml to about 1 mg/ml.

In some embodiments, the transglutaminase reaction is carried out on asolid support. For example, the Fc-containing polypeptide (such asantibody) may be attached to a solid support. The remaining componentsof the conjugation reaction are then brought into contact with theFc-containing polypeptide on the solid support and subsequently removed.Alternatively, the transglutaminase may be attached to a solid support.The remaining components of the conjugation reaction are then broughtinto contact with the transglutaminase on the solid support andsubsequently separated from the transglutaminase on the solid support.

Solid support that are useful for the methods described herein include,for example, plates, tubes, bottles, flasks, magnetic beads, magneticsheets, porous matrices, or any solid surfaces and the like. Agents ormolecules that may be used to link the TGase or Fc-containingpolypeptide to the solid support include, but are not limited to,lectins, avidin/biotin, inorganic or organic linking molecules. Thephysical separation can be effected, for example, by filtration,isolation, magnetic field, centrifugation, washing, etc.

In some embodiments, the solid support is a bead, a membrane, acartridge, a filter, a microtiter plate, a test tube, solid powder, acast or extrusion molded module, a mesh, a fiber, a magnetic particlecomposite, or any other solid materials. The solid support may be coatedwith a substance such as polyethylene, polypropylene,poly(4-methulbutene), polystyrene, polyacrylate, polyethyleneterephthalate, rayon, nylon, poly(vinyl butyrate), polyvinylidenedifluoride (PCDF), silicones, polyformaldehyde, cellulose, celluloseacetate, nitrocellulose, and the like. In some embodiments, the solidsupport may be coated with a ligand or impregnated with the ligand.

In some embodiments, the supporting material is a magnetic bead. In someembodiments, the magnetic beads have an average size of about 1-200microns, such as any of about 1-2 microns, 2-10 microns, 10-30 microns,30-50 microns, 50-100 microns, and 10-200 microns. In some embodiments,the magnetic beads are monodisperse. In some embodiments, the magneticbeads are coated, for example with protein A.

Other solid support that can be used in the methods described hereininclude, but are not limited to, gelatin, glass, sepharose macrobeads,dextran microcarriers such as CYTODES® (Pharmacia, Uppsala, Sweden).Also contemplated are polysaccharide such as agrose, alginate,carrageenan, chitin, cellulose, dextran or starch, polyacrylamide,polystyrene, polyacrolein, polyvinyl alcohol, polymethylacrylate,perfluorocarbon, inorganic compounds such as silica, glass, kieselquhr,alumina, iron oxide or other metal oxides, or copolymers consisting ofany combination of two or more naturally occurring polymers, syntheticpolymers or inorganic compounds.

The amount of the transglutaminase in the reaction mixture (i.e., amountper ml of resin when resin is used as solid support) can be controlledto allow efficient transglutaminase reaction. For example, in someembodiments, the concentration of the tranglutaminase in the reactionmixture (amount per ml of resin) is about any of about 0.01 mg/ml toabout 1 mg/ml, including for example any of about 0.01 mg/ml to about0.02 mg/ml, about 0.02 mg/ml to about 0.03 mg/ml, about 0.03 mg/ml toabout 0.04 mg/ml, about 0.04 mg/ml to about 0.05 mg/ml, about 0.05 mg/mlto about 0.06 mg/ml, about 0.06 mg/ml to about 0.07 mg/ml, about 0.07mg/ml to about 0.08 mg/ml, about 0.08 mg·ml to about 0.09 mg/ml, about0.09 mg/ml to about 0.1 mg/ml, about 0.1 mg/ml to about 0.2 mg/ml, about0.2 mg/ml to about 0.3 mg/ml, about 0.3 mg/ml to about 0.4 mg/ml, about0.4 mg/ml to about 0.5 mg/ml, about 0.5 mg/ml to about 0.6 mg/ml, about0.6 mg/ml to about 0.7 mg/ml, about 0.7 mg/ml to about 0.8 mg/ml, about0.8 mg/ml to about 0.9 mg/ml, about 0.9 mg/ml to about 1 mg/ml.

In some embodiments, the concentration ratio between the conjugatemoiety and the Fc-containing polypeptide (such as antibody) is fromabout 2:1 to about 800:1, including but not limited to about any of:2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 25:1, 30:1,35:1, 40:1, 45:1, 50:1, 60:1, 70:1, 80:1, 90:1, 100:1, 200:1, 300:1,400:1, 500:1, 600:1, 700:1, and 800:1.

In some embodiments, the conjugation efficiency of the Fc-containingpolypeptide (such as antibody) and the conjugation moiety is at leastabout 30%. As used herein, the term “conjugation efficiency” or“crosslinking efficiency” is the ratio between the experimentallymeasured amount of engineered polypeptide conjugate divided by themaximum expected engineered polypeptide conjugate amount. Conjugationefficiency or crosslinking efficiency can be measured by varioustechniques well known to persons skilled in the art, such as hydrophobicinteraction chromatography. Conjugation efficiency can also be measuredat different temperature, such as room temperature or 37° C. In someembodiments, the conjugation efficiency of the Fc-containing polypeptideand the conjugation moiety is at least about any of 30%-35%, 35%-40%,45%-50%, 50%-55%, 56%-60%, 61%-65%, 66%-70%, 71%-75%, 76%-80%, 81%-85%,86%-90%, 91%-95%, or 96%-99%. In some embodiments, the conjugationefficiency of the Fc-containing polypeptide and the conjugation moietyis at least about any of 32%, 34%, 36%, 38%, 40%, 42%, 44%, 46%, 48%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%.

TGases

TGases catalyze covalent protein crosslinking by forming proteinaseresistant isopeptide bonds between a lysine donor residue of one proteinand an acceptor glutamine residue of another protein, and is accompaniedby the release of ammonia. The catalytic mechanism of transglutaminaseshas been proposed as follows. After the glutamine-containing firstsubstrate (acceptor or Q-substrate) binds to the enzyme, it forms agamma-glutamylthioester with the cysteine residue in the active centerof TGase, known as the acylenzyme intermediate, accompanied by therelease of ammonia. The second substrate (donor or K-substrate) thenbinds to the acylenzyme intermediate and attacks the thioester bond. Theproduct (two proteins crosslinked by an Nepsilon (gamma-glutamyl)lysineisopetide bridge) is formed and released. This re-establishes theactive-center Cys residue of the enzyme in its original form and allowsit to participate in another cycle of catalysis. The formation of thecovalent acylenzyme intermediate is thought to be the rate-limiting stepin these reactions. The catalytic triad of many transglutaminases ispapain-like, containing Cys-His-Asp (where His is histidine and Asp isaspartic acid) and, crucially, a tryptophan (Trp) residue located 36residues away from the active-center Cys. In contrast, bacterial TGasesisolated from Streptoverticillium sp (vide supra) has an atypicalcatalytic triad and shows no sequence homology with the papain-likecatalytic triad of other TGases.

Several types of transglutaminases have been reported in various livingorganisms including microbials. Examples are TGase from guinea pig liver(GTGase), fish liver (FTGase) and microorganisms (mTGase) and anyrecombinant TGase (rTGase). Other TGases than the ones listed here canalso be used according to the invention. Examples of useful TGasesinclude microbial transglutaminases, such as e.g. from Streptomycesmobaraense, Streptomyces cinnamoneum and Streptomyces griseocarneumdisclosed in U.S. Pat. No. 5,156,956, and Streptomyces lavendulaedisclosed in U.S. Pat. No. 5,252,469, and Streptomyces ladakanumdisclosed in JP2003199569. Other useful microbial transglutaminases havebeen isolated from Bacillus subtilis (disclosed in U.S. Pat. No.5,731,183) and from various Myxomycetes. Other examples of usefulmicrobial transglutaminases are those disclosed in WO 96/06931 (e.g.transglutaminase from Bacilus lydicus) and WO 96/22366. Usefulnon-microbial transglutaminases include guinea-pig livertransglutaminase, and transglutaminases from various marine sources likethe flat fish Pagrus major (disclosed in EP-0555649), and the Japaneseoyster Crassostrea gigas (disclosed in U.S. Pat. No. 5,736,356). Anexemplary TGase is bacterial transglutaminase (BTG) (see, e.g. EC2.3.2.13, protein-glutamine-gamma-glutamyltransferase). In anotherexemplary embodiment, the TGase is from S. mobaraense. In anotherembodiment, the TGase is a mutant (e.g., engineered) TGase having atleast 80% sequence homology with native TGase. An example is recombinantbacterial transglutaminase derived from Streptomyces mobaraensis(available from Zedira, Darmstadt, Germany).

Streptomyces ladakanum ATCC 27441 or NRRL3191 mTgase is expressed asPre-Pro-mTgase (GenBank access number AY241675). There are 410 aminoacid residues in pre-pro-mTGase, 331 in mature enzyme plus 30 of pre and49 of pro. Pro peptide is a strong inhibitor of mature enzyme. Primersdesigned according to AY241675 were used to clone the pro-mTgase andmature mTgase from ATCC 27441DNA into pET29b(+) vector's Nde I and Xho Isites. Active mTgase can be obtained either from enterokinase lightchain (EKL) digestion of Pro-mTgase or refolding of mature mTgase.mTgase from Strep Ladakanum (TG_SL) is very similar to mTgase fromStrep. mobaraensis (TG_SM, sold by Ajinomoto as ACTIVA®) with a fewamino acid differences (alignment shown in FIG. 2).

The transglutaminase used in methods described herein can be obtained ormade from a variety of sources. In some embodiments, thetransglutaminase is a calcium dependent transglutaminase which requirescalcium to induce enzyme conformational changes and allow enzymeactivity. For example, transglutaminase can be derived from guinea pigliver and obtained through commercial sources (e.g., Sigma-Aldrich (StLouis, Mo.) and MP Biomedicals (Irvine, Calif.)). In some embodiments,the transglutaminase is a calcium independent transglutaminase whichdoes not require calcium to induce enzyme conformational changes andallow enzyme activity. In some embodiments, the transglutaminase is amicrobial transglutaminase derived from a microbial genome, such astransglutaminase from Streptoverticillium or Streptomices (e.g.,Streptomyces mobarensis or Streptoverticillium mobarensis). In someembodiments, the transglutaminase is a mammalian protein (e.g., humantransglutaminase), a bacterial protein, a plant protein, a fungi protein(e.g., Oomycetes and Actinomicetes transglutaminases), or a prokaryoticprotein. In some embodiments, the transglutaminase is from Micrococcus,Clostridium, Turolpsis, Rhizopus, Monascus, or Bacillus.

Suitable TGase include, but is not limited to, bacterialtransglutaminase (BTG) such as the enzyme having EC reference EC2.3.2.13 (protein-glutamin-γ-glutamyltransferase). In some embodiments,the TGase is from Strep Ladakanum (TG_SL, SEQ ID NO:16, see FIG. 2). Insome embodiments, the TGase is from Strep Mobaraensis (TG-SM, SEQ IDNO:18, see FIG. 2). In some embodiments, the TGase is a recombinantTGase based on the TGase from Strep Ladakanum (TG_SL, SEQ ID NO:17, seeFIG. 3).

In some embodiments, the transglutaminase used in the methods describedherein is a recombinant protein produced using recombinant techniques.

In some embodiments, the transglutaminase is wildtype, for example theTGase having the sequence of SEQ ID NO:16. In some embodiments, thetransglutaminase is a recombinant wildtype TGase comprising the wildtypeTGase having the sequence of SEQ ID NO:16, wherein the recombinantwildtype TGase further comprises an additional proline at the N-terminusand optionally a purification tag (such as a polyhistidine tag). In someembodiments, the transglutaminase is a recombinant wildtype TGase havinga sequence of SEQ ID NO:17 as shown in FIG. 3. Contrary to the generalunderstanding in the art that wildtype transglutaminase is unable tocatalyze transglutamination reaction to an acceptor glutamine flanked byan N-glycosylation site, it was surprisingly found that such reactioncan be carried out with substantial efficacy and specificity undercertain conditions as described here.

In some embodiments, the transglutaminase is engineered. In someembodiments, the transglutaminase is an engineered transglutaminasespecifically designed to carry out transglutamination reactions to anacceptor glutamine proximal to an N-glycosylation site. Such engineeredtranglutaminases are further described below in detail.

In some embodiments, the transglutaminase is a purified protein. Forexample, in some embodiments, the transglutaminase is least about 50%pure. As used herein, “pure” or “purified” protein refers to a protein(e.g., transglutaminase) free from other protein contaminants. In someembodiments, the purified transglutaminase is at least about any of55%-60%, 60%-65%, 65%-70%, 70%-75%, 75%-80%, 80%-85%, 85%-90%, 90%-95%,95%-98%, or 99% pure. In some embodiments, the purified transglutaminaseis at least about any of 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 96%, 97%, 98%, 99%, or 100% pure.

Engineered Transglutaminase

The present application in another aspect provides engineeredtransglutaminase specifically designed to carry out transglutaminationreactions to an acceptor glutamine proximal to an N-glycosylation site.We remodeled the substrate binding pocket of TGase to increase theaccessibility of the glutamine residue on Fc region (Q295 specifically)to TGase's catalytic residue Cys64 (FIG. 3), and obtained engineeredTGases that specifically carry out transglutamination reactions at Q295.

In some embodiments, the engineered TGase is based on the wildtype TGasefrom Strep ladalanum (SEQ ID NO:16 or SEQ ID NO:17). In someembodiments, the engineered TGase is based on the wildtype TGase fromStrep Mobaraensis (SEQ ID NO:18). The sequence of a TGase isolated fromStrep ladakanum has an amino acid sequence which is identical to thesequence from Strep mobaraensis except for a total of 22 amino aciddifferences between the two sequences (Yi-Sin Lin et al., ProcessBiochemistry 39(5), 591-598 (2004).

In some embodiments, the engineered transglutaminase specificallycarries out the transglutamination reaction at the acceptor glutaminesite at the N-glycosylated Fc region. The term “specifically” used inthis context describes a preference of the TGase for reacting with oneor more specific glutamine residues at the N-glycosylated Fc region ascompared to other specific glutamine residues on the Fc-containingpolypeptide (such as antibody).

Thus, for example, in some embodiments, there is provided an engineeredtransglutaminase capable of conjugating an Fc-containing polypeptide(such as antibody) to a conjugate moiety, wherein the Fc-containingpolypeptide (such as antibody) comprises an N-glycosylated Fc region,wherein the N-glycosylated Fc region comprises an acceptor glutamineresidue flanked by an N-glycosylation site, wherein upon reaction theconjugate moiety is conjugated to the Fc-containing polypeptide (such asantibody) via the acceptor glutamine residue, and wherein theconjugation is at least about 10% more active than a wildtypetransglutaminase under the same reaction conditions. In someembodiments, the engineered TGase has at least about 80% identity to SEQID NO:16 and further comprises at least one mutation (such assubstitution, deletion, or insertion).

In some embodiments, there is provided an engineered transglutaminasecomprising an amino acid sequence having at least about 80% (includingfor example at least about any of 85%, 90%, 95%, or 95%) identity to SEQID NO:16, wherein the transglutaminase comprises a deletion selectedfrom the group consisting of: D1-E4; P244-P247; and H279-H289. In someembodiments, there is provided an engineered transglutaminase comprisingan amino acid sequence that is 100% identical to SEQ ID NO:16 except forone or more deletions selected from the group consisting of: D1-E4;P244-P247; and H279-H289.

In some embodiments, there is provided an engineered transglutaminasecomprising an amino acid sequence having at least about 80% (includingfor example at least about any of 85%, 90%, 95%, or 95%) identity to SEQID NO:17, wherein the transglutaminase comprises a deletion selectedfrom the group consisting of: P1-E5; P245-P248; and H280-H290. In someembodiments, there is provided an engineered transglutaminase comprisingan amino acid sequence that is 100% identical to SEQ ID NO:17 except forone or more deletions selected from the group consisting of: P1-E5;P245-P248; and H280-H290.

In some embodiments, there is provided an engineered transglutaminasecomprising an amino acid sequence having at least about 80% (includingfor example at least about any of 85%, 90%, 95%, or 95%) identity to SEQID NO:16, wherein the transglutaminase comprises a mutation selectedfrom the group consisting of: deletion of D1-E4; deletion of P244-P247;deletion of N282-L285; substitution of H279-A287 with a G; andsubstitution of A280-H289 with a G. In some embodiments, there isprovided an engineered transglutaminase comprising an amino acidsequence that is 100% identical to SEQ ID NO:16 except for one or moredeletions selected from the group consisting of: a mutation selectedfrom the group consisting of: deletion of D1-E4; deletion of P244-P247;deletion of N282-L285; substitution of H279-A287 with a G; andsubstitution of A280-H289 with a G.

In some embodiments, there is provided an engineered transglutaminasecomprising an amino acid sequence having at least about 80% (includingfor example at least about any of 85%, 90%, 95%, or 95%) identity to SEQID NO:17, wherein the transglutaminase comprises a mutation selectedfrom the group consisting of: deletion of P1-E5; deletion of P245-P248;deletion of N283-L286; substitution of H280-A288 with a G; andsubstitution of A281-H290 with a G. In some embodiments, there isprovided an engineered transglutaminase comprising an amino acidsequence that is 100% identical to SEQ ID NO:17 except for one or moredeletions selected from the group consisting of: a mutation selectedfrom the group consisting of: deletion of P1-E5; deletion of P245-P248;deletion of N283-L286; substitution of H280-A288 with a G; andsubstitution of A281-H290 with a G.

In some embodiments, there is provided an engineered transglutaminasecomprising an amino acid sequence that is 100% identical to SEQ ID NO:16except for a deletion of D1-E4. In some embodiments, there is providedan engineered transglutaminase comprising an amino acid sequence that is100% identical to SEQ ID NO:16 except for a deletion of P244-P247. Insome embodiments, there is provided an engineered transglutaminasecomprising an amino acid sequence that is 100% identical to SEQ ID NO:16except for a deletion of H279-H289. In some embodiments, there isprovided an engineered transglutaminase comprising an amino acidsequence that is 100% identical to SEQ ID NO:16 except for a deletion ofN282-L285. In some embodiments, there is provided an engineeredtransglutaminase comprising an amino acid sequence that is 100%identical to SEQ ID NO:16 except for a deletion of D1-E4 and a deletionof N282-L285. In some embodiments, there is provided an engineeredtransglutaminase comprising an amino acid sequence that is 100%identical to SEQ ID NO:16 except for a deletion of D1-E4, a deletion ofP244-P247, and a deletion of N282-L285. In some embodiments, there isprovided an engineered transglutaminase comprising an amino acidsequence that is 100% identical to SEQ ID NO:16 except for a deletion ofD1-E4 and a substitution of H280-A288 with a G. In some embodiments,there is provided an engineered transglutaminase comprising an aminoacid sequence that is 100% identical to SEQ ID NO:16 except for adeletion of D1-E4 and a substitution of A280-H289 with a G.

In some embodiments, there is provided an engineered transglutaminasecomprising an amino acid sequence that is 100% identical to SEQ ID NO:17except for a deletion of P1-E5. In some embodiments, there is providedan engineered transglutaminase comprising an amino acid sequence that is100% identical to SEQ ID NO:17 except for a deletion of P245-P248. Insome embodiments, there is provided an engineered transglutaminasecomprising an amino acid sequence that is 100% identical to SEQ ID NO:17except for a deletion of H280-H290. In some embodiments, there isprovided an engineered transglutaminase comprising an amino acidsequence that is 100% identical to SEQ ID NO:17 except for a deletion ofN283-N286. In some embodiments, there is provided an engineeredtransglutaminase comprising an amino acid sequence that is 100%identical to SEQ ID NO:17 except for a deletion of P1-E5 and a deletionof N283-N286. In some embodiments, there is provided an engineeredtransglutaminase comprising an amino acid sequence that is 100%identical to SEQ ID NO:17 except for a deletion of P1-E5, a deletion ofP245-P248, and a deletion of N283-N286. In some embodiments, there isprovided an engineered transglutaminase comprising an amino acidsequence that is 100% identical to SEQ ID NO:17 except for a deletion ofP1-E5 and a substitution of H280-A288 with a G. In some embodiments,there is provided an engineered transglutaminase comprising an aminoacid sequence that is 100% identical to SEQ ID NO:17 except for adeletion of P1-E5 and a substitution of A281-H290 with a G.

The terms “sequence identity” or “identify” as used interchangeablyherein refers the degree of sequence relatedness between peptides, asdetermined by the number of matches between strings of two or more aminoacid residues. Sequence identity can be measured, for example, by thepercent of identical matches between the smaller of two or moresequences with gap alignments (if any) addressed by a particularmathematical model or computer program (i.e., “algorithms”). Somemethods to determine identity are designed to give the largest matchbetween the sequences tested. Methods to determine identity aredescribed in publicly available computer programs. Exemplary computerprogram methods to determine identity between two sequences include theGCG program package, including GAP (Devereux et al., Nucl. Acid. Res.12, 387 (1984); Genetics Computer Group, University of Wisconsin,Madison, Wis.), BLASTP, BLASTN, and FASTA (Altschul et al., J. Mol.Biol. 215, 403-410 (1990)). The BLASTX program is publicly availablefrom the National Center for Biotechnology Information (NCBI) and othersources (BLAST Manual, Altschul et al. NCB/NLM/NIH Bethesda, Md. 20894;Altschul et al., supra). The well-known Smith Waterman algorithm mayalso be used to determine identity.

In some embodiments, the engineered transglutaminase has a highertransglutaminase activity than that of the TGase encoded by SEQ ID NO:16or SEQ ID NO:18. In some embodiments, the specificity activity of theengineered transglutaminase is at least about 1.25×, 1.5×, 2.0×, 2.5×,3.0×, 3.5×, 4.0×, 4.5×, 5.0×, 5.5×, 6.0×, 6.5×, 7.0×, 7.5×, 8.0×, 8.5×,9.0×, 9.5×, or 10.5× higher than that of the wildtype TGase (such as theTGase encoded by SEQ ID NO:16 or SEQ ID NO:17).

The engineered TGases described herein can be analyzed for TGaseactivity by using assays known in the art. For example, U.S. Pat. No.5,156,956 describes the measurement of the activity of a given peptideis carried out by performing a reaction usingbenzyloxycarbonyl-L-glutaminyl glycine and hydroxylamine as substratesin the absence of Ca²⁺, forming an iron complex with the resultinghydroxamic acid in the presence of trichloroacetic acid, measuringabsorption at 525 nm and determining the amount of hydroxamic acid by acalibration curve to calculate the activity. For the purpose of thisspecification, a peptide, which exhibits transglutaminase activity insaid assay, is deemed to have transglutaminase activity. In particular,the peptides of the present invention exhibit an activity which is morethan 30%, such as more than 50%, such as more than 70%, such as morethan 90% of that of a TGase from S. ladakanum having an amino acidsequence of SEQ ID No. 16.

Also provided herein are nucleic acids (such as isolated nucleic acids)encoding any one of the engineered TGases described herein. As usedherein the term “nucleic acid” is intended to indicate any nucleic acidmolecule of cDNA, genomic DNA, synthetic DNA or RNA origin. The nucleicacid may be single- or double-stranded, and which may be based on acomplete or partial naturally occurring nucleotide sequence encoding aprotein of interest. The nucleic acid may optionally contain othernucleic acid segments.

Also provided herein are recombinant vectors (such as amplificationvectors and/or expression vectors) comprising nucleic acids encoding theengineered TGases described herein. In some embodiments, there isprovided a host cell comprising the recombinant vector.

The recombinant vector comprising the nucleic acid encoding theengineered TGase may be any vector which may conveniently be subjectedto recombinant DNA procedures, and the choice of vector may depend onthe host cell into which it is to be introduced. Thus, the vector may bean autonomously replicating vector, i.e. a vector which exists as anextrachromosomal entity, the replication of which is independent ofchromosomal replication, e.g. a plasmid. Alternatively, the vector maybe one which, when introduced into a host cell, is integrated into thehost cell genome and replicated together with the chromosome(s) intowhich it has been integrated. The vector is in some embodiments anexpression vector in which the DNA sequence encoding the engineeredTGase is operably linked to additional segments required fortranscription of the DNA. The term, “operably linked” indicates that thesegments are arranged so that they function in concert for theirintended purposes, e.g. transcription initiates in a promoter andproceeds through the DNA sequence coding for the protein. The promotermay be any DNA sequence which shows transcriptional activity in the hostcell of choice and may be derived from genes encoding proteins eitherhomologous or heterologous to the host cell. The DNA sequence encodingthe engineered TGase may also, if necessary, be operably connected to asuitable terminator.

In some embodiments, the recombinant vector further comprises DNAsequence(s) enabling the vector to replicate in the host cell inquestion, and/or a selectable marker, e.g. a gene the product of whichcomplements a defect in the host cell, such as the gene coding fordihydrofolate reductase (DHFR) or the Schizosaccharomyces pombe TPI gene(described by P. R. Russell, Gene 40, 125-130 (1985)), or one whichconfers resistance to a drug, e.g. ampicillin, kanamycin, tetracyclin,chloramphenicol, neomycin, hygromycin or methotrexate. For filamentousfungi, selectable markers include amdS, pyrG, argB, niaD and sC.

The host cell into which the vector comprising a nucleic acid encodingthe engineered TGase is introduced may be any cell which is capable ofproducing the engineered TGase and includes bacteria, yeast, fungi andhigher eukaryotic cells. The transformed or transfected host celldescribed above is then cultured in a suitable nutrient medium underconditions permitting the expression of the present peptide, after whichthe resulting protein is recovered from the culture. In someembodiments, the host cell is a prokaryotic cell. In some embodiments,the host cell is S. ladakanum. In some embodiments, the host cell is S.mobaraensis. In some embodiments, the host cell is E. coli.

Further provided herein are methods of preparing the engineered TGasesdescribed herein. The engineered TGases described herein may be preparedin different ways. For example, in some embodiments, the engineeredTGase is prepared by culturing a host cell comprising a vectorcomprising a nucleic acid encoding the engineered TGase and isolatingthe engineered TGase from the cells.

To direct a protein of the present invention into the secretory pathwayof the host cells, a secretory signal sequence (also known as a leadersequence, prepro sequence or pre sequence) may be provided in therecombinant vector. The secretory signal sequence is joined to the DNAsequence encoding the protein in the correct reading frame. Secretorysignal sequences are commonly positioned 5′ to the DNA sequence encodingthe protein. The secretory signal sequence may be that normallyassociated with the protein or may be from a gene encoding anothersecreted protein. Alternatively, the protein may be expressed in theinclusion body, and subsequently obtained throughdenaturation/renaturation.

The medium used to culture the cells may be any conventional mediumsuitable for growing the host cells, such as minimal or complex mediacontaining appropriate supplements. The protein produced by the cellsmay then be recovered from the culture medium by conventional proceduresincluding separating the host cells from the medium by centrifugation orfiltration, precipitating the proteinaceous components of thesupernatant or filtrate by means of a salt, e.g. ammonium sulphate,purification by a variety of chromatographic procedures, e.g. ionexchange chromatography, gel filtration chromatography, affinitychromatography, or the like, dependent on the type of protein inquestion.

In some embodiments, there are provided methods of purifying TGase, suchas any one of the TGase described herein. In some embodiments, themethod comprises (a) providing a host cell that expresses TGase; (b)culturing said host cell (such as a prokaryotic cell) wherein TGase isexpressed as an inclusion body; (c) disrupting said host cell to producea cell lysate having a soluble fraction and an insoluble fraction; and(d) separating said soluble fraction from said insoluble fraction,wherein said insoluble fraction comprises the TGase. In someembodiments, the method further comprises contacting the insolublefraction comprising TGase in a denaturing agent (such as urea). In someembodiments, the method further comprises containing the denaturingTGase to a renaturation buffer (such as a buffer comprising DTT). Insome embodiments, the method further comprises purifying the TGase bychromatography (such as by affinity chromatography or ion exchangechromatography). In some embodiments, the TGase is tagged (such ashis-tagged) to facilitate purification.

In some embodiments, there is provided a method of purifying TGase,comprising (a) culturing a host cell (such as a prokaryotic cell)comprising a vector comprising a nucleic acid encoding a pro-enzyme ofTGase, and (b) obtaining mature TGase by cleavage of the pro-sequence ofthe pro-enzyme (for example by endokinase light chain).

The mutant TGases described herein can be used for making Fc-containingpolypeptide conjugates (such as antibody drug conjugates). For example,in some embodiments, there is provided a method of making an antibodydrug conjugate comprising an antibody specifically conjugated to aconjugate moiety, comprising: contacting the antibody with the conjugatemoiety in the presence of a mutant transglutaminase (such as any of themutant transglutaminase described herein) under a condition that issufficient to generate the antibody drug conjugate, wherein theconjugate moiety is conjugated to an acceptor glutamine residue on theantibody. In some embodiments, there is provided a method of making anantibody drug conjugate comprising an antibody specifically conjugatedto a conjugate moiety comprising: contacting the antibody with theconjugate moiety in the presence of a mutant transglutaminase (such asany of the mutant transglutaminase described herein) under a conditionthat is sufficient to generate the antibody drug conjugate, wherein theantibody is glycosylated (e.g., N-glycosylated) in the Fc-region, andwherein the conjugate moiety is conjugated to the endogenous acceptorglutamine residue on the antibody. In some embodiments, there isprovided a method of making an antibody drug conjugate comprising anantibody specifically conjugated to a conjugate moiety comprising:contacting a composition comprising the antibody with the conjugatemoiety in the presence of a mutant transglutaminase (such as any of themutant transglutaminase described herein) under a condition that issufficient to generate the Fc-containing polypeptide conjugate, whereinat least some (e.g., 50%, 60%, 70%, 80%, 90%, or more) the antibody inthe composition is glycosylated (e.g., N-glycosylated) in the Fc-region,and wherein the conjugate moiety is conjugated to the endogenousacceptor glutamine residue on the antibody.

In some embodiments, there is provided a method of making an antibodydrug conjugate comprising an antibody specifically conjugated to aconjugate moiety comprising a small molecule handle and an active moietycomprising: a) contacting the antibody with the small molecule handle inthe presence of a transglutaminase (such as any of the mutanttransglutaminase described herein) under a condition that is sufficientto generate an intermediate conjugate comprising an antibodyspecifically conjugated to the small molecule handle, and b) contactingthe intermediate conjugate with an active moiety thereby obtaining theantibody drug conjugate, wherein the conjugate moiety is conjugated toan acceptor glutamine residue on the antibody. In some embodiments,there is provided a method of making an antibody drug conjugatecomprising an antibody specifically conjugated to a conjugate moietycomprising a small molecule handle and an active moiety comprising: a)contacting the antibody with the small molecule handle in the presenceof a transglutaminase (such as any of the mutant transglutaminasedescribed herein) under a condition that is sufficient to generate anintermediate conjugate comprising an antibody specifically conjugated tothe small molecule handle, and b) contacting the intermediate conjugatewith an active moiety thereby obtaining the antibody drug conjugate,wherein the antibody is glycosylated (e.g., N-glycosylated) in theFc-region, and wherein the conjugate moiety is conjugated to theendogenous acceptor glutamine residue on the antibody. In someembodiments, there is provided a method of making an antibody drugconjugate comprising antibody specifically conjugated to a conjugatemoiety comprising a small molecule handle and an active moietycomprising: a) contacting a composition comprising antibody with thesmall molecule handle in the presence of a transglutaminase (such as anyof the mutant transglutaminase described herein) under a condition thatis sufficient to generate an intermediate conjugate comprising anantibody specifically conjugated to the small molecule handle, and b)contacting the intermediate conjugate with an active moiety therebyobtaining the antibody drug conjugate, wherein at least some (e.g., 50%,60%, 70%, 80%, 90%, or more) the antibody in the composition isglycosylated (e.g., N-glycosylated) in the Fc-region, and wherein theconjugate moiety is conjugated to the endogenous acceptor glutamineresidue on the antibody.

Pharmaceutical Compositions, Unit Doses, and Kits

Also provided are pharmaceutical compositions comprising theFc-containing polypeptide conjugates (such as antibody drug conjugates)described herein. In some embodiments, the pharmaceutical compositionfurther comprises a pharmaceutically acceptable carrier. In someembodiments, at least about 50% (such as at least about any of 60%, 70%,80%, 90%, 95%, or 99%) of the Fc-containing polypeptide conjugates (suchas antibody drug conjugates) in the pharmaceutical composition has oneconjugate moiety attached to the Fc-containing polypeptide (such asantibody). In some embodiments, at least about 50% (such as at leastabout any of 60%, 70%, 80%, 90%, 95%, or 99%) of the Fc-containingpolypeptide conjugates (such as antibody drug conjugates) in thepharmaceutical composition has two conjugate moieties attached to theFc-containing polypeptide (such as antibody). In some embodiments, atleast about 50% (such as at least about any of 60%, 70%, 80%, 90%, 95%,or 99%) of the Fc-containing polypeptide conjugates (such as antibodydrug conjugate) in the pharmaceutical composition has either one or twoconjugate moieties attached to the Fc-containing polypeptide (such asantibody).

The term “pharmaceutically acceptable carrier” is used herein todescribe any ingredient other than the compound(s) of the invention. Thechoice of excipient(s) to a large extent depend on factors such as theparticular mode of administration, the effect of the excipient onsolubility and stability, and the nature of the dosage form. As usedherein, “pharmaceutically acceptable carrier” includes any and allsolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like that arephysiologically compatible. Some examples of pharmaceutically acceptableexcipients are water, saline, phosphate buffered saline, dextrose,glycerol, ethanol and the like, as well as combinations thereof. In someembodiments, isotonic agents, including, but not limited to, sugars,polyalcohols (e.g., mannitol, sorbitol) or sodium chloride are includedin the pharmaceutical composition. Additional examples ofpharmaceutically acceptable substances include, but are not limited to,wetting agents or minor amounts of auxiliary substances such as wettingor emulsifying agents, preservatives or buffers, which enhance the shelflife or effectiveness of the antibody.

In some embodiments, the Fc-containing polypeptide conjugates (such asantibody drug conjugates) described herein can be deimmunized to reduceimmunogenicity upon administration to a subject suing known techniquessuch as those described, e.g., in PCT Publication WO98/52976 andWO00/34317.

The pharmaceutical compositions described herein may be prepared,packaged, or sold in bulk, as a single unit dose, or as a plurality ofsingle unit doses. As used herein, a “unit dose” is discrete amount ofthe pharmaceutical composition comprising a predetermined amount of theactive ingredient. The amount of the active ingredient is generallyequal to the dosage of the active ingredient which would be administeredto a subject or a convenient fraction of such a dosage such as, forexample, one-half or one-third of such a dosage. Any method foradministering peptides, proteins or antibodies accepted in the art maysuitably be employed for the Fc-containing polypeptide conjugatesdisclosed herein.

The pharmaceutical compositions described herein in some embodiments aresuitable for parenteral administration. Parenteral administration of apharmaceutical composition includes any route of administrationcharacterized by physical breaching of a tissue of a subject andadministration of the pharmaceutical composition through the breach inthe tissue, thus generally resulting in the direct administration intothe blood stream, into muscle, or into an internal organ. For example,parenteral administration includes, but is not limited to,administration of a pharmaceutical composition by injection of thecomposition, by application of the composition through a surgicalincision, by application of the composition through a tissue-penetratingnon-surgical wound, and the like. In particular, parenteraladministration is contemplated to include, but is not limited to,subcutaneous, intraperitoneal, intramuscular, intrasternal, intravenous,intraarterial, intrathecal, intraventricular, intraurethral,intracranial, intrasynovial injection or infusions; and kidney dialyticinfusion techniques. In some embodiments, parenteral administration isthe intravenous or the subcutaneous route.

Formulations of a pharmaceutical composition suitable for parenteraladministration may be prepared, packaged, or sold in a form suitable forbolus administration or for continuous administration. Injectableformulations may be prepared, packaged, or sold in unit dosage form,such as in ampoules or in multi dose containers containing apreservative. Formulations for parenteral administration include, butare not limited to, suspensions, solutions, emulsions in oily or aqueousvehicles, pastes, and the like. Such formulations may further compriseone or more additional ingredients including, but not limited to,suspending, stabilizing, or dispersing agents. In one embodiment of aformulation for parenteral administration, the active ingredient isprovided in dry (i.e. powder or granular) form for reconstitution with asuitable vehicle (e.g. sterile pyrogen free water) prior to parenteraladministration of the reconstituted composition. Parenteral formulationsalso include aqueous solutions which may contain excipients such assalts, carbohydrates and buffering agents (preferably to a pH of from 3to 9), but, for some applications, they may be more suitably formulatedas a sterile non-aqueous solution or as a dried form to be used inconjunction with a suitable vehicle such as sterile, pyrogen-free water.Exemplary parenteral administration forms include solutions orsuspensions in sterile aqueous solutions, for example, aqueous propyleneglycol or dextrose solutions. Such dosage forms can be suitablybuffered, if desired. Other parentally-administrable formulations whichare useful include those which comprise the active ingredient inmicrocrystalline form, or in a liposomal preparation. Formulations forparenteral administration may be formulated to be immediate and/orengineered release. Engineered release formulations include controlled,delayed, sustained, pulsed, targeted and programmed releaseformulations. For example, in one aspect, sterile injectable solutionscan be prepared by incorporating the Fc-containing polypeptideconjugate, e.g., antibody-drug conjugate or bispecific antibody-drugconjugate, in the required amount in an appropriate solvent with one ora combination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the exemplary methods of preparation arevacuum drying and freeze drying that yields a powder of the activeingredient plus any additional desired ingredient from a previouslysterile filtered solution thereof. The proper fluidity of a solution canbe maintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. Prolonged absorption of injectablecompositions can be brought about by including in the composition anagent that delays absorption, for example, monostearate salts andgelatin.

An exemplary, non-limiting pharmaceutical composition of theFc-containing polypeptide conjugate (such as antibody drug conjugate) isa formulation as a sterile aqueous solution having a pH that ranges fromabout 5.0 to about 6.5 and comprising from about 1 mg/mL to about 200mg/mL of an engineered polypeptide conjugate disclosed herein, fromabout 1 millimolar to about 100 millimolar of histidine buffer, fromabout 0.01 mg/mL to about 10 mg/mL of polysorbate 80, from about 100millimolar to about 400 millimolar of trehalose, and from about 0.01millimolar to about 1.0 millimolar of disodium EDTA dehydrate.

Dosage regimens may be adjusted to provide the optimum desired response.For example, a single bolus may be administered, several divided dosesmay be administered over time or the dose may be proportionally reducedor increased as indicated by the exigencies of the therapeuticsituation. It is especially advantageous to formulate parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form, as used herein, refers tophysically discrete units suited as unitary dosages for thepatients/subjects to be treated; each unit containing a predeterminedquantity of active compound calculated to produce the desiredtherapeutic effect in association with the required pharmaceuticalcarrier. The specification for the dosage unit forms of the inventionare generally dictated by and directly dependent on (a) the uniquecharacteristics of the agent moiety (e.g., small molecules such ascytotoxic agent) and the particular therapeutic or prophylactic effectto be achieved, and (b) the limitations inherent in the art ofcompounding such an active compound for the treatment of sensitivity inindividuals.

The skilled artisan would appreciate, based upon the disclosure providedherein, that the dose and dosing regimen is adjusted in accordance withmethods well-known in the therapeutic arts. That is, the maximumtolerable dose can be readily established, and the effective amountproviding a detectable therapeutic benefit to a patient may also bedetermined, as can the temporal requirements for administering eachagent to provide a detectable therapeutic benefit to the patient.Accordingly, while certain dose and administration regimens areexemplified herein, these examples in no way limit the dose andadministration regimen that may be provided to a patient in practicingthe present invention.

It is to be noted that dosage values may vary with the type and severityof the condition to be alleviated, and may include single or multipledoses. It is to be further understood that for any particular subject,specific dosage regimens should be adjusted over time according to theindividual need and the professional judgment of the personadministering or supervising the administration of the compositions, andthat dosage ranges set forth herein are exemplary only and are notintended to limit the scope or practice of the claimed composition.Further, the dosage regimen with the compositions of this invention maybe based on a variety of factors, including the type of disease, theage, weight, sex, medical condition of the patient, the severity of thecondition, the route of administration, and the particular antibodyemployed. Thus, the dosage regimen can vary widely, but can bedetermined routinely using standard methods. For example, doses may beadjusted based on pharmacokinetic or pharmacodynamic parameters, whichmay include clinical effects such as toxic effects and/or laboratoryvalues. Thus, the present invention encompasses intra-patientdose-escalation as determined by the skilled artisan. Determiningappropriate dosages and regimens are well-known in the relevant art andwould be understood to be encompassed by the skilled artisan onceprovided the teachings disclosed herein.

The invention also provides kits (or articles of manufacture) for use inthe treatment of the disorders described above. Kits of the inventioninclude one or more containers comprising an Fc-containing polypeptideconjugate (such as antibody drug conjugate) for treating a disease. Forexample, the instructions comprise a description of administration ofthe engineered Fc-containing polypeptide conjugate (such as antibodydrug conjugate) to treat a disease, such as cancer (e.g., pancreatic,ovarian, colon, breast, prostate, or lung cancer). The kit may furthercomprise a description of selecting an individual suitable for treatmentbased on identifying whether that individual has the disease and thestage of the disease. The instructions relating to the use of theengineered Fc-containing polypeptide conjugate (such as antibody drugconjugate) generally include information as to dosage, dosing schedule,and route of administration for the intended treatment. The containersmay be unit doses, bulk packages (e.g., multi-dose packages) or sub-unitdoses. Instructions supplied in the kits of the invention are typicallywritten instructions on a label or package insert (e.g., a paper sheetincluded in the kit), but machine-readable instructions (e.g.,instructions carried on a magnetic or optical storage disk) are alsoacceptable. The kits of this invention are in suitable packaging.Suitable packaging includes, but is not limited to, vials, bottles,jars, flexible packaging (e.g., sealed Mylar or plastic bags), and thelike. Also contemplated are packages for use in combination with aspecific device, such as an inhaler, nasal administration device (e.g.,an atomizer) or an infusion device such as a minipump. A kit may have asterile access port (for example the container may be an intravenoussolution bag or a vial having a stopper pierceable by a hypodermicinjection needle). The container may also have a sterile access port(for example the container may be an intravenous solution bag or a vialhaving a stopper pierceable by a hypodermic injection needle). At leastone active agent in the composition is an engineered polypeptide asdescribed herein. The container may further comprise a secondpharmaceutically active agent. The kits may optionally provideadditional components such as buffers and interpretive information.Normally, the kit comprises a container and a label or package insert(s)on or associated with the container.

In some embodiments, there is provided a kit comprising a TGase (such asan engineered TGase, such as any one of the engineered TGase describedherein). In some embodiments, the kit further comprises other reagentsfor carrying out the tranglutamination reaction. In some embodiments,the kit further comprises an instruction on carrying out any one of theconjugation methods described herein. In some embodiments, the kitfurther comprises a solid support for immobilizing the TGase (such asthe engineered TGase) or the Fc-containing polypeptide (such asantibody). In some embodiments, the TGase (such as the engineered TGase)in the kit is immobilized on the solid support.

EXAMPLES

The invention can be further understood by reference to the followingexamples, which are provided by way of illustration and are not meant tobe limiting.

Example 1. Generation of TGase Mutants

This example describes the generation of TGase mutants. With referenceto FIG. 3, three regions which near active site entrance were deleted ormutated to enlarge the substrate binding pocket. Wild type TG_SL wascloned into pET39+ vector using NdeI and XhoI with an extra proline (SEQID NO:17). The following deletion mutants were made based on IgG1 andTG_SM docking: Mutant 1: Deletion from P1-E5 (SEQ ID NO:37); Mutant 2:Deletion from P245-P248 (SEQ ID NO:38); Mutant 3: Deletion fromN283-L286 (SEQ ID NO:39); Mutant 4: Deletions of P1-E5 and N283-L286(SEQ ID NO:40); Mutant 5: Deletion of all three regions specified bymutants 1, 2 and 3 (SEQ ID NO:41); Mutant 6: Deletion of P1-E5 andreplace H280-A288 with G (SEQ ID NO:42); Mutant 7: Deletion of P1-E5 andreplace A281-H290 with G (SEQ ID NO:43). Deletion area is greyed out,underlined or stricken-out as shown in FIG. 3. These mutants were moreactive toward IgG1.

Using DNA purified from S. ladakanum (ATCC27441) as PCR template, DNAsequences coding for wild type pro- or mature mTGase (wildtype TG_SL)and its mutants 1-3 (see above) were cloned into the pET39b vector atthe NdeI and BamHI sites. Inclusion bodies of mature wildtype TG_SL andmutants 1-3 were obtained from E. coli BL21 (DE3) cells transformed withrespective vectors. After solubilization in 8 M urea, wildtype TG_SL andmutants were refolded by dilution into renaturation buffer (1 mM DTT, 50mM Tris, pH 8.0). The enzymes were further purified by Ni-NTA and cationexchange columns. Alternatively, Pro-mTgase was expressed as solubleinactive pro-enzyme in E. coli. Then, the active enzyme was obtainedafter cleavage of the pro domain by endokinase light chain (EKL).

Example 2. Conjugation of IgG1 with Monodansylcadaverine (MDC) Catalyzedby mTgase

MDC was chosen for this experiment because it has a primary amine andits fluorescence can be easily monitored. MDC is used here todemonstrate its conjugation to mAB. To purified IgG1 (1-10 mg/ml) inTris-buffer (pH 6.5-8.5), add MDC (Sigma-Aldrich) in DMSO to finalconcentrations of 1-5 mM (final DMSO 2-10%). Add purified wildtype TG_SLor its mutant to a final concentration of 0.05-1.0 mg/ml. Incubate thereaction mixtures at 37° C. Reaction was followed by HPLC using phenylhydrophobic interaction column (PHIC, Tosoh Bioscience LLC). At thebeginning of the reaction, the product was dominated by DAR1, where onlyone heavy chain of IgG1 was coupled with MDC. As the reactionprogressed, DAR 2, where both heavy chains of IgG1 are coupled with MDC,became the major product. Toward the end of reaction (8 hours at 0.2mg/ml of mTgase at pH 7), conjugation yield reached 80% for DAR2 with20% of DAR 1 left, or 90% for heavy chain (HC) when the sample wasreduced by 10 mM TCEP and analyzed on C4-1000A column (Vydac) (see FIG.6). The selective conjugation of MDC to HC was visualized on SDS PAGE(FIG. 7). Other mTgases, such as TG_SM from S. mobaraensis (purifiedfrom Ajinomoto's Activa TI), was also tested. Mutants were more activethan the wild type toward IgG1, although wild type TGase could alsocatalyze ADC reaction at high concentration (>0.1 mg/ml). It was furtherfound that TG_SM (sold by Ajinomoto and used by Pfizer and InnatePharma) also works at high concentration, but only had about 30%activity comparing to TG_SL.

Example 3. Pegylation of IgG1 with 1 kDa mPEG-NH2 by mTgase Catalysis

This experiment was carried out essentially as described in Example 2.The acyl acceptor MDC was replaced with 1 kDa methoxy-PEG-amine (JenKem,USA) in pH 7.0 to a final concentration of 1 to 2 mM, PEGylated IgG1 wasobtained. Sample analysis of an overnight reaction at 37° C. on a C4column after reduction with TCEP showed 90% modification of the heavychains.

Example 4. Conjugation of IgG1 with Monodansylcadaverine (MDC) Catalyzedby Immobilized mTgase

To simplify mTgase removal and allow reuse of the enzyme, immobilizedmTgase was used in catalysis. In preparing a column of immobilizedmTgase, 1 ml of 15 mg/ml of mTgase in carbonate buffer (pH 8.3) was usedfor each NHS activated HITRAP® HP column of 1.0 ml (GE) followingmanufacturer's protocol. 0.5 ml of purified IgG1 at 1-10 mg/ml inTris-buffer (pH 6-8.0) with 1-5 mM of MDC was injected intoHITRAP®-mTgase column. The column was sealed at both ends and incubatedat 37° C. overnight. The next day, reaction mixture was eluted with Trisbuffer. The column was rejuvenated with 1-20 mM TCEP for the nextconjugation reaction. There was no loss of activity of immobilizedmTgase after each use. Yield of 90% HC was reached at each run, similarto the yield obtained with free mTgase.

Example 5. Conjugation of IgG1, 2 and 4 with Cytotoxins Catalyzed bymTgase

Toxins with an amine linker could be conjugated to IgG1 in a One-Stepprotocol just as MDC does (FIG. 4). Although a linker as simple as—(CH2)_(n)—NH2 (where n≥4 as in lysine side chain), use of ethyleneglycol scaffold could increase the solubility of linker-drug andfacilitate the conjugation reactions. This example demonstratesconjugation of MAY-PEG4 (non-cleavable linker, FIG. 8) and MAY-PVCL(cleavable linker, FIG. 9) to IgG1.

A maytansine derivative containing an extended, non-cleavable linear PEGlinker with a primary amine group of MW: 896.42 Da is depicted in FIG.8. MAY-PEG4 in DMSO was added to IgG1 (1-10 mg/ml in pH 8.0 Tris buffer)to a final concentration of 1-2 mM. mTgase was added to a finalconcentration of 0.2-1.0 mg/ml and the reactions were incubated at 37°C. The reaction was monitored by HPLC analysis as described in example2. After overnight, a yield of 60% modified heavy chains was obtained.Both DAR 1 and DAR 2 products were seen (FIG. 10).

A maytansine derivative containing a cleavable linker with aself-immolative spacer and terminal lysine of molecular weight 1224.58Da is shown in FIG. 9. Conjugation reaction was run the same way asabove by replacing MAY-PEG4 with MAY-PVCL (1.0 mg/ml). After incubationat 37° C. for 8 hours, 40% of the heavy chain was modified (FIG. 11).Low yield is attributed to the low solubility of the drug.

Example 6. Drug to Antibody Ratio (DAR) Determination and ConjugationSite Mapping on IgG1

Due to the heterogeneity of glycan chains, IgG1 would display multiplepeaks on its Mass Spectra. To simplify mass analysis, all mAB conjugateswere deglycoslyated before mass spectrometer analysis using PNGaseF(Promega, Madison, Wis.), so a single peak would be observed for eachspecies of the same charge. By doing so, the original glycan linkedasparagine (N) is changed to aspartate (D).

Mass Confirmation of DAR1 and DAR2. Expected DAR 1 and DAR 2 ofIgG1-MAY-PEG4 from Example 5 were purified on Phenyl HIC. Afterdeglycosylation, samples were spotted on a 196 well steel plate andanalyzed on MALDI-TOF (ABI 4700, Applied Biosystems, Redwood City,Calif.). As a control, naked IgG1 was used (DAR0). Mass spectra wereacquired in positive High Mass linear mode and multiple charged species(double and triple) were used to calculate molecular weight. MAY-PEG4drug has a molecular weight of 896 Da. Therefore conjugation of onemolecules of MAY-PEG4 to IgG1 (DAR1) would result in expected massdifference of 879 Da (896−17 loss of NH3=879 Da), whereas conjugation oftwo molecules to IgG1 (DAR2) would result in mass difference of 1758 Da.MALDI-TOF spectra in FIG. 10 confirmed DAR1 and DAR2.

Confirmation of Conjugation on Heavy Chain Only. To confirm that drugmolecule was conjugated to IgG1's heavy chain (HC) but not light chain(LC), purified DAR2 of IgG1-MAY-PVCL from Example 5 was deglycosylatedand reduced with 20 mM DTT for 30 min at 37° C. Mass spectra wereacquired in positive High Mass linear mode using ABI4700. MAY-PVCL drughas a molecular weight of 1224 Da. Therefore, conjugation of oneMAY-PVCL to heavy chain would result in expected mass difference of 1207Da (1224−17=1207 Da) and no difference in molecular weight of lightchains (FIG. 11). On the other hand, DAR1 has both naked HC andHC-MAY-PVCL peaks on its mass spectrum, indicating DAR1 has only one HCconjugated.

Peptide Mapping to Verify Site-Specific Conjugation at Q295. PurifiedDAR2 from Example 2 (both heavy chains of IgG1 containing MDC) and nakedIgG1 were deglycosylated, reduced, alkylated, digested into peptidesusing trypsin and/or chymotrypsin (Promega, Madison, Wis.) and separatedby reversed phase chromatography (C18) prior to mass spectrometryanalysis. Digested peptides were monitored on HPLC by UV absorbance at328 nm (λmax of MDC). Only one peak at 328 nm was identified in DAR2samples, whereas no peak was detected in control antibody. MALDI-TOFanalysis identified that peak as a single charged peptide EEQYDSTYR (SEQID NO:27) from trypsin digestion or NAKTKPREEQY (SEQ ID NO:28) fromchymotrypsin digestion containing Glutamine 298 (Sequential Q298 and isQ295 by Kabat numbering system) with exactly one MDC (1508.7observed-1190.5 peptide+335 MDC-17 NH3=318 Da; 1681.9 observed-1363.6peptide=318) (gray rows in Table 1). To exclude other Glutamines (otherthan Q298) as additional conjugation sites, full peptide mappingexperiments were performed using unmodified IgG1 and IgG1-MDC conjugate.The digested samples were directly analyzed without purification toidentify all glutamine-containing peptides on heavy chain. Out of all 16glutamines, Q298 was the only conjugation site with MDC attached(Table 1) while all other glutamine containing peptides remainunchanged.

TABLE 1 Glutamine containing peptides identified after proteasesdigestion* Glutamine (Q) IgG1 Peptides from Trypsin or PeptideSequential Expected Observed IgG1-MDC Chymotrypsin* Digestion PositionNumbering Mass Mass Observed Mass EVQLVESGGGLVQPGGSLR   1-13 Q3, Q131882.2 1882.1 1882.1 (SEQ ID NO: 20) VESGGGLQPGGSL*   5-18 Q13 1256.61256.7 1256.7 (SEQ ID NO: 21) QAPGKGLEWVAR  39-50 Q39 1311.7 1311.61311.7 (SEQ ID NO: 22) NTAYLQMNSLR  77-87 Q82 1310.6 1310.6 1310.7(SEQ ID NO: 23) WGGDGFYAMDYWGQGTLVTVSSASTK  99-124 Q112 2784.2 2784.22784.2 (SEQ ID NO: 24) TSGVHTFPAVLOSSGL* 167-182 Q178 1600.8 1600.91600.9 (SEQ ID NO: 25) GTQTY* 197-201 Q199  569.2  569.3  569.3(SEQ ID NO: 26) EEQYDSTYR 296-304 Q298 1190.5 1190.5 1508.7 (SEQ ID NO: 27) (1190.5 + 318) NAKTKPREEQY* 288-299 Q298 1363.5 1363.61681.9 (SEQ ID NO: 28) (1363.6 + 318) EVQLVESGGGLVQPGGSLR 305-320 Q3141808.0 1808.0 1808.0 (SEQ ID NO: 29) GQPREPQVYTLPPSR 344-358 Q345 1724.91724.8 1724.9 (SEQ ID NO: 30) EPQVYTLPPSR 348-358 Q350 1286.6 1286.51286.6 (SEQ ID NO: 31) KNQVSLTCLVK 364-373 Q365 1104.6 1105.6 1105.6(SEQ ID NO: 32) GFYPSDIAVEWESNGQPENNYK 374-395 Q389 2544.1 2544.5 2544.5(SEQ ID NO: 33) TVDKSRWQQGNVF* 414-426 Q421 1564.7 1564.7 1564.8(SEQ ID NO: 34) WQQGNVFSCSVMHEALHNHYTQK 420-442 Q421, Q422, 2744.22744.7 2744.8 (SEQ ID NO: 35) Q441 *Note: Sequential Q298 and is Q295 byKabat numbering system. N300 (or Kabat N297) became D300 whendeglycosylated.

To confirm Q298 as the specific conjugation site on IgG1 when realcytotoxins were used, IgG1 conjugates of MAY-PEG4 and TAM1 (a tubulysinA derivative with an amine linker) were deglycosylated, reduced,alkylated, digested into peptides using trypsin and separated byreversed phase chromatography prior to mass spectrometry analysis. Thesame peptide EEQYDSTYR (SEQ ID NO:27) containing Q298 was identified inboth IgG1-TAM1 and IgG1-MAY-PEG4 conjugates with mass corresponding toone drug molecule attached: 2134.0 (1190.5+960.5−17) and 2069.8(1190.5+895.5−17), respectively (Table 2).

TABLE 2 Q298-containing peptide identified after tryptic digest IgG1IgG1-TAM1 Peptide Glutamine Expected Observed Observed IgG1-MAY-PEG4Peptide Position (Q) Mass Mass Mass Observed Mass EEQYDSTYR 296-304 Q2981190.5 1190.5 2134.0 2069.8 (SEQ ID (1190.5 + (1190.5 + NO: 27)960.5 - 17) 895.5 - 17)

Example 7. Conjugation of IgG Subclasses Catalyzed by mTGase

Purified human IgG 2 or IgG 4 at 1-10 mg/ml in Tris buffer (pH 7.0-8.0)was reacted with 2-5 mM of MDC following the addition of 0.1 to 1 mg/mLof purified mTgase. The mixture was incubated at 37° C. for 8-16 hoursand then analyzed on phenyl hydrophobic interaction column or reduced by10 mM TCEP on C4 column. Similar to IgG1, IgG2 and IgG4 were conjugatedwith MDC to show DAR1 and DAR2 accumulating with time (FIG. 6).

Example 8. Two-Step Protocol to Prepare Antibody Drug Conjugates UsingmTGase

While the one step conjugation reaction is simple and straightforward,the yield is affected by the solubility of the drug. When drugconcentration is low, the by-product from deamidation may besignificant. To suppress deamidation, a highly soluble amine containingchemical handle was used in excess (molar ratio of chemical:mAB>10) inthe first step conjugation catalyzed by mTgase. Then, drug moleculeswere cross-linked to mAB in the second step via chemoselective ligationreactions (FIG. 5). Many chemoselective pairs can be used:

Amino-oxy-/Aldehyde or Ketone

Sulfhydryl/Maleimide

Azide/alkyne

IgG1 conjugation of PEG by mTgase via amino propyl acetal. To 1-10 mg/mlof IgG1 in pH 7.0-8.0 Tris buffer, add 3-aminopropionaldehyde diethylacetal (CAS#41365-75-7) to a final concentration of 2-50 mM and mTgaseto 0.05 to 0.5 mg/ml. The reaction mixture was incubated at 37° C. for2-16 hours until reaction reached completion. After diafiltration toremove excess acetal, adjust the pH to 2-4 for 2-10 hours at roomtemperature with formic acid or HCl to regenerate aldehyde group. AdjustpH of IgG1-aldehyde with sodium carbonate back to 5-8. Add amino-oxy-PEG(20 kDa) to 3 to 4 times of IgG1 (molar ratio) plus a catalyst of 50 to100 mM aniline or 10 mM of 5-methoxyanthranilic acid. After overnightincubation at room temperature, IgG1-(PEG20k)₂ reached yield of 95%.

IgG1 conjugation of drug by mTgase via amine-azide. To 1-10 mg/ml ofIgG1 in pH 7.0-8.0 Tris buffer, add 3-azido-1-propanamine(CAS#88192-19-2) to a final concentration of 2-50 mM and mTgase to 0.05to 0.5 mg/ml. The reaction mixture was incubated at 37° C. for 2-16hours. Yield reached 100%. After diafiltration to remove excess azidopropyl amine, DBCO-Maytansine was added to 3 times of IgG (by molar).IgG1-(Maytansine)₂ yield reached over 95%.

Example 9. In Vitro Cell Assay to Assess ADC Potency Prepared by mTgase

SK-BR-3 cells were seeded in 96 well black clear-bottom plates at 10Kcells/well and cultured for 24 hours. Cells were treated for 96 hourswith 2 fold serially diluted antibody-drug conjugates in triplicates.Cells viability was determined by CELLTITER™ Blue Cell Viability Assay(Promega, Madison, Wis.). Relative cell viability was determined as apercentage of untreated control. IC50 was calculated using a fourparameter logistic model from XLfit. Table 3 shows the drug to antibodyratio and IC50 in SK-BR-3 cells using various trastuzumab-drugconjugates.

TABLE 3 IC50 of antibody-drug conjugates in SK-BR-3 cells IC₅₀ IC₅₀ DrugDAR (μg/ml ADC) (nM drug equivalent) ADC-TAM1 DAR1 0.033 0.22 ADC-TAM1DAR2 0.017 0.22 ADC-MAY-PEG4 DAR1 0.046 0.31 ADC-MAY-PEG4 DAR2 0.0280.38 ADC MAY-PVCL DAR1 0.081 0.54 ADC MAY-PVCL DAR2 0.055 0.72

Example 10. Site-Specific ADCs Prepared by mTgase with StableNon-Cleavable Linkers are Highly Stable and Potent in Xenograft Mice

Trastuzumab (10 mg/ml) and Monomethyl auristatin E (MMAE) with each of 9non-cleavable PEG linkers (CH₂CH₂O)_(x) (x=2, 4, 6, 8, 10, 12, 16, 20,and 24, FIG. 12) were conjugated respectively as described in Example 5.ADCs were purified by Protein A column to remove mTgase and excess MMAE.The average DARs are ˜1.9 when the reduced ADC samples were analyzed onHPLC using C4 column. In SK-BR-3 cell based assay, these ADCs are allpotent with IC50 from 38 to 148 pM (Table 4).

TABLE 4 IC50 (pM) of Trastuzumab-MMAE conjugates in SK-BR-3 orBT474cells Linker IC₅₀ SK-BR-3 IC₅₀ BT474 PEG2 148 1095 PEG4 61 273 PEG650 234 PEG8 42 230 PEG10 40 271 PEG12 38 267 PEG16 40 281 PEG20 72 495PEG24 114 807 PEG3c 60 283

Six of the trastuzumab-MMAE conjugates were selected for in vivo testusing BT474 xenograft mice. Each female athymic nude mouse (4 week old,18-22 g; Harlan) was implanted with one estrogen 3 mm (60 day slowrelease, Innovative Research of America) tablet 2-3 days prior to cellinjection. BT474 cells were resuspended to a final 50-60 millioncells/ml, and mixed 1:1 with matrigel, then 200 μl was injectedsubcutaneously into the flank of each mouse. Treatment starts when thetumor volume (½×L×W×H) reaches around 200 mm³ after 3 weeks. ADCs werediluted into PBS buffer to a final concentration 1 mg/ml and wereadministered intravenously into mice tails at about 200 μl to reach 10mg/kg dose. Tumor size was measured daily using a digital caliper. Eventhough Linker PEG6 and 8 seemed to be optimal in the BT474 cell-basedassay (Table 4), the difference in in vivo efficacy is very small (FIG.13).

Since all 6 ADCs were potent, we only tested one ADC with PEG12 linkerfor its stability in blood. NCI N87 xenograft mice were generated in asimilar way as the BT474 xenograft mice with ˜5 million cells per mouseexcept no estrogen tablet was used. After ADC administration, bloodsamples of 20 μl were taken every 1-2 days up to 21 days by poking mousetails and mixed with 120 μl of storage buffer (PBS with 10 mM EDTA and0.1 M NH4Cl). Then total trastuzumab and ADC were analyzed by sandwichELISA. Black NUNC® Maxisorp 96 plates were coated with Her2 protein at100 ng/well. Samples were further diluted with PBS as needed to suit thelinear detection range of 10 pg to 2 ng (for either Trastuzumab or ADC).After applying samples (fresh ADC dilutions were used as both total mAband ADC standards) and washing, rabbit polyclonal anti-trastuzumab (fortotal mAb) or anti-MMAE (for total ADC) were applied as secondaryantibodies while Goat anti-Rabbit IgG-HRP (Life-technologies) was usedas the detection antibody. AMPLEX® Red (Cayman Chemical)/4-Iodophenol(Sigma)/H₂O₂ mixture was used as fluorescence substrate. Plates wereread on SpectraMax GEMINI™ with 555 nm Ex and 585 nm Em. The ratio ofADC/mAb vs time was plotted in FIG. 14. It is clear that site-specificADC with a stable non-cleavable linker is completely stable in blood.

Example 11. DAR 2 Site-Specific ADC Prepared by mTgase with a CleavableLinker is More Stable and Potent than Commercial TDM-1 in XenograftModels

Trastuzumab (10 mg/ml) and MMAE with cleavable PEG3c linker (FIG. 12)was conjugated and purified as described in Example 10. This ADC, namedas TP3cE, has DAR of 1.9, and high potency in vitro (Table 4). In vivostudies were conducted in comparison to TDM-1 (Genentech) in both NCIN87 and SK_Ov3 xenograft models. In NCI N87 model, TP3cE is 4 times moreefficacious than TDM-1 with a single intravenous injection (FIG. 15).Blood sample analysis show that TP3cE is completely stable in blood forup to 21 days while TDM-1 lost 50% of its toxin in 5 days (FIG. 16). InSK_Ov3 xenografts, TP3cE at 3 weekly doses of either 15 or 8 mg/kgresulted in complete tumor remission while TDM-1 showed efficacy only at15 mg/kg (FIG. 17).

Example 12. DAR 4 Site-Specific ADC Prepared by mTgase with Two-StepProcess

Trastuzumab (10 mg/ml) and each of a group of 3-arm PEG linkers (1-5kDa) with one amine group and two azide groups (FIG. 18, top;Conju-probe and Jenkem) (4-8 mg/ml) were conjugated and purifiedrespectively as described in Example 10. The antibody-linker conjugationreactions reached >90% conversion when analyzed reduced by HPLC using aC4 column.

Five-fold molar excess of Alkyne-PEG4c-MMAE (FIG. 18, bottom panel) wasthen coupled to one of the products above, trastuzumab-3-arm-PEG (1 kDa)(1-10 mg/ml), in the presence of 0.1-1 mM CuSO₄ and 1-5 mM Sodiumascorbate for 10-300 minutes. The final DAR 4 ADC product, denotedTP6TP4cE, was then purified by protein A as described in Example 10 andthe actual DAR was 3.8 as determined by HPLC using a C4 column. TP6TP4cEin vitro activity is higher than TP3cE of DAR 2 as shown in Table 5.

TABLE 5 IC50 (pM) of Trastuzumab-MMAE conjugates in BT474cells LinkerDAR IC₅₀ BT474 TP3cE 2 280 TP6TP4cE 3.8 80

The invention claimed is:
 1. A method of making an antibody drugconjugate comprising an antibody specifically conjugated to a conjugatemoiety comprising: contacting an antibody composition comprising theantibody with the conjugate moiety in the presence of a transglutaminaseunder a condition sufficient to generate the antibody drug conjugate,wherein at least about 50% of the antibody in the antibody compositionis glycosylated in the Fc-region, wherein the conjugate moiety isconjugated to an endogenous acceptor glutamine residue on the antibody,and wherein the transglutaminase is a wildtype transglutaminase and theconcentration of the transglutaminase in the reaction mixture is about0.1 mg/ml to about 1 mg/ml.
 2. The method of claim 1, wherein thetransglutaminase is derived from Streptomyces ladakanum or Streptomycesmobaraensis.
 3. The method of claim 1, wherein the transglutaminaseconsists of the amino acid sequence of SEQ ID NO: 16, or SEQ ID NO: 18.4. The method of claim 1, wherein the transglutaminase has a purity ofat least about 90%.
 5. The method of claim 1, wherein the molar ratio ofthe transglutaminase and the antibody composition is about 10:1 to about1:10.
 6. The method of claim 1, wherein the transglutaminase isimmobilized on a solid support.
 7. The method of claim 1, wherein theantibody is immobilized on a solid support.
 8. The method of claim 1,where the antibody is a human or humanized antibody.
 9. The method ofclaim 1, wherein both heavy chains of the antibody are conjugated to theconjugate moiety.
 10. The method of claim 1, wherein the conjugatemoiety comprises an active moiety selected from the group consisting ofa moiety that improves the pharmacokinetic property of the antibodycomposition, a therapeutic moiety, and a diagnostic moiety.
 11. Themethod of claim 1, wherein the conjugate moiety comprises a toxin. 12.The method of claim 1, wherein the antibody is N-glycosylated in theFc-region.
 13. The method of claim 1, wherein the concentration ratiobetween the conjugate moiety and the antibody is from about 2:1 to about800:1.
 14. A method of making an antibody drug conjugate comprising anantibody specifically conjugated to a conjugate moiety comprising asmall molecule handle and an active moiety comprising: a) contacting anantibody composition comprising the antibody with the small moleculehandle in the presence of a transglutaminase under a conditionsufficient to generate an intermediate conjugate comprising the antibodyspecifically conjugated to the small molecule handle, wherein thetransglutaminase is a wildtype transglutaminase and the concentration ofthe transglutaminase in the reaction mixture is about 0.1 mg/ml to about1 mg/ml, and b) contacting the intermediate conjugate with the activemoiety thereby obtaining the antibody drug conjugate, wherein at leastabout 50% of the antibody in the antibody composition is glycosylated inthe Fc-region, wherein the conjugate moiety is conjugated to anendogenous acceptor glutamine residue on the antibody, and wherein theactive moiety is selected from the group consisting of a moiety thatimproves the pharmacokinetic property of the antibody composition, atherapeutic moiety, and a diagnostic moiety.
 15. The method of claim 14,wherein the small molecule handle has the structure of NH₂—R, wherein Ris a moiety that allows the attachment of the active moiety.
 16. Amethod of making an antibody drug conjugate comprising an antibodyspecifically conjugated to a conjugate moiety comprising: contacting anantibody composition comprising the antibody with the conjugate moietyin the presence of a transglutaminase under a condition sufficient togenerate the antibody drug conjugate, wherein at least about 50% of theantibody in the antibody composition is glycosylated in the Fc-region,wherein the conjugate moiety is conjugated to an endogenous acceptorglutamine residue on the antibody, and wherein the transglutaminase is awildtype transglutaminase with an added proline residue at theN-terminus, and optionally a purification tag, and the concentration ofthe transglutaminase in the reaction mixture is about 0.1 mg/ml to about1 mg/ml.